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B Ehrenberg

Publications and source records attributed to B Ehrenberg.

At least 19 recordsLinked to original sources
PubMed2003

Specificity of photosensitizer accumulation in undifferentiated versus differentiated colon carcinoma cells.

The relationship between tumor cell differentiation and photosensitizer accumulation used in PDT is poorly defined. In the present work, specific cell differentiation of colon carcinoma CT26 cells induced by sodium butyrate was manifested by morphological changes, proliferation and protein expression and was correlated with the accumulation of endogenous and exogenous photosensitizes. Reduced accumulation of the endogenous protoporphyrin IX and the exogenous hypericin and MC540 was detected in differentiated cells. In contrast, a differentiation-dependent increase was measured with TPPS4, TMPyP, the pheophorbides (C5, C6, C12), HypS4 and helianthrone. In conclusion, PpIX, Hypericin and MC540 show specific binding and accumulation in poorly differentiated tumors, giving these tumors tissue-specific advantage in photo-diagnostic PDT applications.

Animals↗
PubMed2001

Spectral imaging of MC540 during murine and human colon carcinoma cell differentiation.

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147-153, 2001)

Alkaline Phosphatase↗
PubMed2001

Solvatochromic effects in the electronic absorption and nuclear magnetic resonance spectra of hypericin in organic solvents and in lipid bilayers.

The natural product hypericin was tested in recent years as a biological photosensitizer with a potential for viral and cellular photodamage. We thus studied extensively its spectroscopy and membrane partitioning. Absorption, fluorescence excitation and emission spectra of the sodium salt (HyNa) were measured in 36 protic and aprotic, polar and apolar, solvents. Electronic transition bands as well as vibrational progressions were identified. Aggregation in some nonpolar solvents and protonation in organic acids were demonstrated. Modeling solvatochromism was done by Lippert equation, by the ET(30) parameter and by the Taft multiparameter approach. In all cases, separation into protic and aprotic solvents gave much better fits to the models. 13C chemical shift data could also be correlated with solvent polarity. They correlated best with Lippert's delta f polarity measure, but tended to fall into two distinct solvent groups--each along different lines--corresponding to protic and aprotic media, respectively. This interesting phenomenon suggests that in the case of the charged and slightly water soluble HyNa, two mechanisms of solvation are involved, each resulting in its own line equation. In aprotic media, dipole-dipole interaction is the predominant solvation mechanism. In protic solvents, the most effective means of solvation is likely to be hydrogen bonding. When intercalated into the liposomal phospholipid bilayer, HyNa is oriented at an angle to the interface, thus experiencing a gradient of solvent polarities: a highly polar environment (similar to methanol) for C-2/5, suggesting that they lie not far from the interface; a moderately polar environment (similar to that of n-propanol) for C-6a/14a, which are somewhat deeper within the bilayer; and a more lipophilic environment (akin to n-hexanol) for C-10/11. The fluorescence excitation peak in liposomes also correlates with an aprotic medium of relatively high polarity, as might be excepted from a molecule in a shallow position in the bilayer.

Anthracenes↗
PubMed2001

Spectroscopic probing of the acid-base properties and photosensitization of a fluorinated phthalocyanine in organic solutions and liposomes.

A perfluorinated derivative of phthalocyanine was synthesized as the free base, hexadeca-(2,2,2-trifluoroethoxy) phthalocyanine (H2F48Pc), and as a zinc complex, hexadeca-(2,2,2-trifluoroethoxy)-phthalocyaninatozinc (ZnF48Pc), and their spectroscopic and photochemical properties were studied. The absorption bands are shifted bathochromically relative to simple phthalocyanines, exhibiting the longest wavelength band near 735 nm (H2F48Pc) and 705 (ZnF48Pc). The solvatochromism of both compounds was modeled by Reichardt's ET(30) parameter and Kamlet, Abboud and Taft multiparameter approach. The former, simpler, model was found to be adequate. We found that H2F48Pc undergoes unique basic and acidic titrations in organic solvents. These titration processes are accompanied by spectral changes that are explained on the basis of the chromophore's symmetry. Singular value decomposition was employed to resolve the spectra into the contributions of the species at various stages of protonation and to obtain the equilibrium constants. Nuclear magnetic resonance spectra (1H, 19F and 13C) for the free base were obtained in a tetrahydrofurand8 solution. The carbon spectrum, taken as a function of temperature, provided evidence for the presence of a tautomerization process, which switches the two internal hydrogens between the four central nitrogen atoms. As far as we know, this is the first report of the measurement of the free energy of activation for such process (delta G = 10.6-11.4 kcal mol-1 between 217 and 330 K) for a phthalocyanine, in solution. Like most other phthalocyanines these two compounds also act as photosensitizers and as generators of singlet molecular oxygen. The absolute quantum yields (phi delta) for ZnF48Pc was 0.58 +/- 0.01 in benzene and 0.35 +/- 0.01 in lipid vesicles. H2F48Pc had lower yields, 0.16 and 0.005, respectively. Either protonation or deprotonation of the pyrrole nitrogens in H2F48Pc lowered the phi delta.

Hydrogen-Ion Concentration↗
PubMed2000

Liposome binding constants and singlet oxygen quantum yields of hypericin, tetrahydroxy helianthrone and their derivatives: studies in organic solutions and in liposomes.

The spectroscopy and photophysics of several hypericin and helianthrone derivatives were studied in methanol and when bound to liposomes. The singlet oxygen quantum yields (phi(delta)) were measured indirectly relative to Rose Bengal and hematoporphyrin IX, employing 9,10-dimethylanthracene as a singlet oxygen trap. Hypericin was found to have a phi(delta) of 0.39+/-0.01 in methanol, and 0.35+/-0.05 in lecithin vesicles, in agreement with literature values. A heavy atom effect was evident upon bromination, resulting in phi(delta) for tetrabromohypericin of 0.72+/-0.02, presumably due to enhanced intersystem crossing. Elimination of the anionic hydroxyls by methylation also enhanced phi(delta) to 0.81+/-0.01. Conversely, addition of anionic sulfate groups drastically reduced phi(delta) resulting in phi(delta)'s of 0.12+/-0.01, 0.052+/-0.003 and 0.40+/-0.01 for hypericin disulfonate, hypericin tetrasulfonate and hexamethyl hypericin tetrasulfonate, respectively. The non-sulfonated helianthrones exhibited low phi(delta)'s in solution. The liposome binding constants, Kb, were measured using a spectroscopic assay. Except for hexamethyl hypericin, all non-sulfonated compounds bound well with Kb's ranging from 15.5+/-0.1 to 48.7+/-3.9 (mg/ml)(-1). None of the tetrasulfonated compounds bound, however the hypericin disulfonate had a Kb of 4.1+/-0.2 (mg/ml)(-1). The phi(delta)'s of the compounds capable of binding were measured and, in the case of the hypericin derivatives, were found not to vary dramatically from those in the free state. Liposome-bound helianthrone and dimethyl tetrahydroxy helianthrone both exhibited high phi(delta)'s, i.e. >0.5. The variations in binding constant and sensitization efficiencies are explained in conjunction with the molecular structure. The relevance of the above data to photodynamic therapy is briefly discussed.

Anthracenes↗
PubMed2000

Mitochondrial localization and photodamage during photodynamic therapy with tetraphenylporphines.

The subcellular localization sites of TPPS4 and TPPS1 and the subsequent cellular site damage during photodynamic therapy were investigated in CT-26 colon carcinoma cells using spectroscopic and electron microscopy techniques. The association of both porphyrins with the mitochondria was investigated and the implications of this association on cellular functions were determined. Spectrofluorescence measurements showed that TPPS4 favors an aqueous environment, while TPPS1 interacts with lipophilic complexes. The subcellular localization sites of each sensitizer were determined using spectral imaging. Mitochondrial-CFP transfected cells treated with porphyrins revealed localization of TPPS1 in the peri-nuclear region, while TPPS4 localized in the mitochondria, inducing structural damage and swelling upon irradiation, as shown by transmission electron microscopy. TPPS4 fluorescence was detected in isolated mitochondria following irradiation. The photodamage induced a 38% reduction in mitochondrial activity, a 30% decrease in cellular ATP and a reduction in Na(+)/K(+)-ATPase activity. As a result, cytosolic concentrations of Na(+) and Ca(2+) increased, and the level of K(+) decreased. In contrast, the lipophilic TPPS1 did not affect mitochondrial structure or function and ATP content remained unchanged. We conclude that TPPS4 induces mitochondrial structural and functional photodamage resulting in an altered cytoplasmic ion concentration, while TPPS1 has no effect on the mitochondria.

Adenocarcinoma↗
PubMed2000

Principal component analysis of the absorption and resonance Raman spectra of the metallochromic indicator antipyrylazo III.

Metallochromic indicators, whose spectral properties are changed in the presence of metal cations, are used mainly in biological studies to monitor Ca2+ and Mg2+ ions. Antipyrylazo III is such indicator, employed for mid-range Ca2+ concentrations (10-1000 microM). The stoichiometry of the interactions of antipyrylazo III with Ca2+, Mg2+, Ba2+, Sr2+ and Zn2+ ions and the relevant binding constants were studied by principal component analysis (PCA) of the absorption spectral changes. The resonance Raman spectra of the above systems were measured as well, and the resolved Raman spectra of the various species were calculated and assigned. The vibrational spectra are more featured, more characteristic of the binding ions and exhibit stronger relative spectral changes upon binding the cations. The basis sets of Raman spectra could thus be used as an analytical tool for these divalent metallic cations.

Barium↗
PubMed2000

Importance of sleep restoration in co-morbid disease: effect of anticonvulsants.

Over the past two to three decades, sleep medicine has emerged as an important discipline as it strives to meet the challenges of some of the most prevalent disorders among humans. Among the 110 disorders listed in the International Classification of Sleep Disorders, two of the most prevalent and treatable have only recently begun to receive significant attention: sleep apnea and restless legs syndrome with sleep-related periodic limb movements disorder. It is becoming clear that the sleep disruption caused by such disorders has ramifications beyond the usually associated daytime sleepiness, and may include: exacerbation of seizures, headaches, short-term memory deficits, and other cognitive problems. Sleep apnea has also been correlated with hypertension and cardiovascular/cerebrovascular disease. Animal studies have taken this one step further by demonstrating that total sleep deprivation is consistently fatal, usually within 1 month, although the precise mechanism remains to be discovered. The most compelling finding in the animal studies is that "rescuing" the animals with sleep, before the irreversible stage, is associated with rebound amounts of deep sleep and rapid eye movement (REM) sleep ("dream sleep"). This same response is seen after initiating treatment of sleep apnea with nasal continuous positive airway pressure (CPAP), and can also occur in patients with other sleep disorders in response to particular medications, such as valproate or gabapentin.

Anticonvulsants↗
PubMed1998

Kinetics and yield of singlet oxygen photosensitized by hypericin in organic and biological media.

The spectroscopy and photophysics of the photosensitizer hypericin when in homogeneous solutions and when bound to liposomes were studied. Hypericin was found to partition efficiently into DMPC liposomes, with a binding constant of 58 (mg lipid/mL)-1. In these liposomes the singlet oxygen production quantum yield was 0.43 +/- 0.09. To determine the deactivation constant of singlet oxygen in lipid bilayers for the first time, we calculated extrapolated values from its quenching by DMPC and lecithin in homogeneous solutions and obtained decay times of 36.4 and 12.2 microseconds, respectively. We also measured the quenching of singlet oxygen, sensitized by hypericin in DMPC liposomes, by NaN3, diphenyl isobenzofuran and H2O:D2O mixtures and explained the results on the basis of singlet oxygen diffusing rapidly out of the lipid bilayer into the aqueous medium. The observed temperature effect on the lifetime of singlet oxygen of about 50% over a 15 degrees C range in liposome suspension contrasts with a 3% change in a homogeneous solution in 1-nonanol and is explained by the temperature effect on the diffusion out of the liposome. A strong pH effect was observed, indicating that the deprotonated species formed above about pH 10 is a much weaker photosensitizer of singlet oxygen than the native, protonated species.

Anthracenes↗
PubMed1997

In vivo photodynamic therapy with the new near-IR absorbing water soluble photosensitizer lutetium texaphyrin and a high intensity pulsed light delivery system.

An in vivo fluorescence monitoring and photodynamic therapy (PDT) study was performed using the new photosensitizer lutetium texaphyrin (Lu-Tex). This photosensitizer is water soluble and has the additional advantage of strong absorption near 730 nm. C26 colon carcinoma was transplanted in the foot of BALB/c mice. In vivo fluorescence spectroscopy was applied to study Lu-Tex tissue distribution kinetics. For this purpose, fluorescence intensity both in the foot with the tumor and in the normal foot was measured in vivo by the laser-induced fluorescence (LIF) system. For PDT, both feet of the mice were irradiated simultaneously with the use of a new high intensity pulsed light delivery system, the Photodyne. The results of the LIF measurements showed that the maximal fluorescence intensity ratio between the normal and tumor bearing foot (FIR) was observed 24-48 h after the agent injection. Photoirradiation with doses from 90 to 240 J cm-2 (0.6 J cm-2 per 2 ms pulse, 1 Hz) 24 h after injection of Lu-Tex at a dose of 10 mg kg-1 caused significant tumor necrosis and delay in the tumor growth rate. The antitumor effect was enhanced with increasing light doses. Normal tissue response to PDT with Lu-Tex was determined as the damage index of the normal foot, which was irradiated simultaneously with the tumor bearing foot. The normal tissue response after PDT with Lu-Tex was compared with 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PP), chlorin e6 (Chl) and Photofrin (PII) at the same values of antitumor effect. The results showed that at 50, 80 and 100% inhibition of tumor growth the orders of the values of normal foot damage indexes were as follows: ALA > Lu-Tex > or = PII > Chl, PII > ALA > Lu-Tex > Chl and PII > Lu-Tex > ALA > Chl respectively.

Animals↗
PubMed1996

A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model.

An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed.

Aminolevulinic Acid↗
PubMed1995

Topical application of 5-aminolevulinic acid, DMSO and EDTA: protoporphyrin IX accumulation in skin and tumours of mice.

Topical 5-aminolevulinic acid (ALA) application in three different creams was carried out on mice bearing subcutaneously transplanted C26 colon carcinoma. The creams contained (a) 20% ALA alone, (b) ALA with 2% dimethylsulphoxide (DMSO) and (c) ALA, DMSO and 2% edetic acid disodium salt (EDTA). Protoporphyrin IX (PP) production in the tumour and in the skin overlying the tumour was studied by two methods: laser-induced fluorescence (LIF) and chemical extraction. The kinetics of PP production in the skin and in the tumour, as studied by the LIF method, was similar for all three cream preparations. The PP fluorescence intensity in the tissues reached its maximum 4-6 h after application of the creams. Quantitative analysis showed that the PP concentration after treatment was more pronounced in the skin than in the tumour. The efficiency of porphyrin production in the skin by the creams used was in the following order: ALA-DMSO-EDTA > ALA-DMSO > ALA. In the tumour the enhancing effect of DMSO and EDTA on PP accumulation induced by ALA was observed mainly in the upper 2 mm section. However, the concentration of PP in the tumour was found to be approximately the same for ALA-DMSO and ALA-DMSO-EDTA cream combinations. The possible mechanisms of the effect of DMSO and EDTA are discussed.

Administration, Topical↗
PubMed1994

Spectroscopy and photosensitization of sapphyrins in solutions and biological membranes.

A spectroscopic and photophysical study of three new sapphyrin molecules is presented. The sapphyrin backbone that was derivatized to make them water soluble possesses an absorption band around 700 nm, a desired property for biological photosensitization. We studied the absorption and fluorescence spectra, from which evidence for aggregation in solvents of different polarities was obtained. The extent of aggregation is correlated with the nature of the attached moiety. The absolute quantum yields of singlet oxygen production were measured, with 1,3-diphenyl isobenzofuran as a model target, and were 0.13-0.18 in ethanol. The binding constants to liposomes and to cells were determined spectroscopically and were found to correspond to the hydrophobicities of the compounds, with an additional effect, ascribed to the sugar moiety, which was found in the case of one of the sapphyrins. The efficiency of photodamage to Staphylococcus aureus by sapphyrins and hematoporphyrin was equivalent, on the basis of cells killed per microgram of sensitizer in the incubation mixture.

Escherichia coli↗
PubMed1994

Epilepsy and sleep apnea syndrome.

We identified seven patients with refractory partial epilepsy and sleep apnea. Treatment of the sleep apnea with nasal continuous positive airway pressure (CPAP), protriptyline, trazodone, acetazolamide, or tracheostomy reduced seizure frequency and severity in six patients. Success with CPAP depended largely on compliance. Four of five patients had a clear reduction in seizure frequency with the use of CPAP. Sleep apnea may exacerbate epilepsy by causing sleep disruption and deprivation, hypoxemia, and decreased cerebral blood flow. In epilepsy patients with risk factors (eg, obesity) or markers (eg, habitual snoring, daytime somnolence) for sleep apnea, a careful sleep history should be elicited and a polysomnogram obtained when indicated. Treatment of the sleep disorder can improve seizure control.

Adult↗
PubMed1993

Electric depolarization of photosensitized cells: lipid vs. protein alterations.

We have monitored several photosensitized reactions in proteins, liposomes and cells under similar conditions. We found that the depolarization of K(+)-diffusion potential of liposomes or the leakage of an entrapped molecule, calcein, progress at a much slower rate than the photosensitized damage to proteins and the photosensitized killing of bacterial and leukemic cells. X-ray microanalysis revealed that upon light exposure of HP-treated leukemic cells and bacteria, they totally lost their cellular potassium. We deduce that the direct photosensitized oxidation of lipid components cannot cause the depolarization of cells, which in turn could be responsible for their death. A photosensitized damage to protein sites in the cell, probably in the membrane, is a more likely reason for the depolarization, the loss of potassium ions and cell death that is caused in light-activated photodynamic processes.

Bacteriorhodopsins↗
PubMed1993

Singlet oxygen generation by porphyrins and the kinetics of 9,10-dimethylanthracene photosensitization in liposomes.

Two new sensitizers are introduced for a potential use in photodynamic therapy: Zn(2+)- and MG(2+)-tetrabenzoporphyrin (ZnTBP and MgTBP). A comparative study of the quantum yields of singlet oxygen generation (phi delta) of hematoporphyrin derivative (HpD), Photofrin II (PF-II), Zn(2+)-phthalocyanine tetrahydroxyl [ZnPC(OH)4] and the newly introduced sensitizers ZnTBP and MgTBP in liposomes, as well as the kinetics of a photochemical reaction sensitized by them, was made by employing the fluorescent membrane probe 9,10-dimethylanthracene (DMA). We followed the photosensitization of DMA in real time by monitoring its fluorescence decrease at 457 nm and found that DMA's photosensitization is oxygen mediated. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the phi delta values were evaluated. At 10 microM sensitizer in an aqueous suspension of 2 mg/mL egg phosphatidylcholine (EPC), HpD was found to have the largest value of phi delta (0.215), followed by PF-II (0.191), ZnTBP (0.023), MgTBP (0.019) and ZnPC(OH)4 (0.005). As a test of the method, phi delta for methylene blue in ethanol was measured and found to be 0.45 as compared to 0.52 reported in the literature. Due to difference in the sensitizers' absorbances at the laser's wavelength, the reaction photosensitized by ZnTBP was the fastest with a time constant of 6.7 min, followed by MgTBP (8.7), PF-II (11.9), HpD (17.1) and ZnPC(OH)4 (31.2), all at equal sensitizers' concentrations and laser intensities. The binding constants of the sensitizers to EPC liposomes are also reported.

Anthracenes↗
PubMed1992

Inactivation of gram-negative bacteria by photosensitized porphyrins.

Photosensitization of Escherichia coli and Pseudomonas aeruginosa cells by deuteroporphyrin (DP) is shown to be possible in the presence of the polycationic agent polymyxin nonapeptide (PMNP). Previous studies established complete resistance of Gram-negative bacteria to the photodynamic effects of porphyrins. The present results show that combined treatment of E. coli or P. aeruginosa cultures with DP and PMNP inhibit cell growth and viability. No antibacterial activity of PMNP alone could be demonstrated and cell viability remained unchanged. Spectroscopically, PMNP was found to bind DP, a mechanism which probably assists its penetration into the cell's membranes. Insertion of DP into the cells was monitored by the characteristic fluorescence band of bound DP at 622 nm. Binding times were 5-40 min and the extent of binding increased with decreasing the pH from 8.5 to 6.5. DP binding constants, as well as the concentrations of PMNP which were required for maximal effect on the various Gram-negative bacteria, were determined fluorometrically. By the treatment of DP, PMNP and light the growth of E. coli and P. aeruginosa cultures was stopped and the viability of the culture was dramatically reduced. Within 60 min of treatment the survival fraction of E. coli culture was 9 x 10(-6) and that of P. aeruginosa was 5.2 x 10(-4). Electron microscopy depicted ultrastructural alterations in the Gram-negative cells treated by DP and PMNP. The completion of cell division was inhibited and the chromosomal domain was altered markedly.

Cell Wall↗