ON THE SOURCE OF THE OSCILLATIONS OBSERVED DURING in vivo ZINC PHTHALOCYANINE FLUORESCENCE PHARMACOKINETIC MEASUREMENTS IN MICE

Surface-detected fluorescence spectroscopy can be used to monitor the pharmacokinetics of uptake and clearance of red-absorbing fluorophores such as zinc(II) phthalocyanine (ZnPc) in vivo. When this technique is applied to mice that have been fed on a normal chlorophyll-based diet, and particularly when measurements are performed in the abdominal region, oscillations are sometimes observed superimposed on the pharmacokinetic curve of the ZnPc. An oscillatory signal has also been observed arising from the abdominal region of control mice fed a normal diet but not injected with the ZnPc photosensitizer; this oscillatory component to the signal is reduced when mice are fed a chlorophyll-free diet. The oscillatory signal component has been attributed to fluorescence arising from chlorophyll derivatives (pheophorbide/pheophytin) contained in the rodent food, whose concentration in the measured abdominal region changes substantially with time, presumably due to digestive processes. Thus it is important to be aware of the possibility of such artifactual contributions to in vivo fluorescence pharmacokinetic measurements.     

ALUMINUM SULFONATED PHTHALOCYANINE DISTRIBUTION IN RODENT TUMORS OF THE COLON, BRAIN and PANCREAS

Abstract The most widely discussed aspect of photodynamic therapy (PDT) is the preferential uptake and retention of sensitisers by malignant tissues. The sensitiser usually used is hematoporphyrin derivative (HPD) but this compound is not an ideal photosensitiser for this purpose and we have therefore studied an aluminum sulfonated phthalocyanine (AlSPc) as a possible alternative. Here we have studied the uptake and retention of this compound in a rat colon cancer, a hamster pancreatic cancer and a mouse glioma, using an alkali extraction technique to estimate tissue AlSPc and comparing the results with those from the corresponding normal tissues. All of the tumors studied reached accumulation peaks at 24-48 h after intravenous administration of AlSPc compared with peaks at 1-3 h in the normal tissues. The tumors outside the central nervous system (CNS) reached peak tumor : normal tissue ratios of 2-3 : 1 and the tumor within the CNS, the malignant glioma, reached a far higher ratio of 28 : 1. These ratios are similar to those reported by others using HPD.     

IN VIVO FLUORESCENCE OF TUMORS AFTER TREATMENT WITH DERIVATIVES OF HEMATOPORPHYRIN

Abstract— Administration of a mixture of porphyrins termed HPD (hematoporphyrin derivative) to mice bearing the Lewis lung tumor leads to preferential accumulation of fluorescence at tumor loci in vivo after 48 h. HPLC analysis shows that the fluorescent species consist of hematoporphyrin and its dehydration products. But injection of these porphyrins does not lead to fluorescence localization. The intracellular fluorescence which is observed apparently arises from intracellular degradation of the tumor-localizing component of HPD. These fluorescent species represent only a small fraction of the total accumulated porphyrin pool; a larger weakly-fluorescent porphyrin pool is also present, and may be the major factor in tumor photosensitization.     

TIME-GATED FLUORESCENCE IMAGING FOR THE DIAGNOSIS OF TUMORS IN A MURINE MODEL

A system for time-gated fluorescence imaging was used to perform measurements on tumor-bearing mice treated with hematoporphyrin derivative (HpD). The aim of the study was to define the potential of this technique in the diagnosis of tumors by taking advantage of the long fluorescence lifetime of the exogenous dye with respect to the decay times of the natural fluorescence. After the administration of three different drug doses (5, 10 and 25 mg/kg body weight), fluorescence images were acquired at various uptake times (from 2 h to 10 d), to determine the best instrumental conditions and experimental procedure for the detection of tumors in the murine model considered. The optimal fluorescence contrast between the tumor area and the surrounding healthy tissue was found at 12 h after the administration of either 5 or 10 mg/kg HpD and was anticipated at 8 h for the highest drug dose. In this optimum condition, the tumor region could be identified even after the injection of 5 mg/kg HpD. A better fluorescence contrast was always obtained in 15 ns-delayed images with respect to synchronous ones.     

A COMPARISON OF FLUORESCENCE METHODS USED IN THE PHARMACOKINETIC STUDIES OF Zn(II)PHTHALOCYANINE IN MICE

The pharmacokinetics of Zn phthalocyanine (ZnPc) encapsulated in dipalmitoyl-phosphatidylcholine (DPPC) liposomes, injected intravenously in Skh:HR-1 nude mice, was monitored by two in vitro techniques and one in vivo technique, all based on fluorescence spectroscopy. The in vitro methods involve either fluorescence measurements on thin tissue sections or on extracts from these tissues. The in vivo method involves the fluorescence measurement at the skin surface. Both in vitro techniques gave similar results which are consistent with previous findings on the pharmacokinetic behavior of ZnPc. The liver and spleen showed rapid ZnPc concentration increases, reaching a maximum level in 30 min. or less post drug administration. Relatively little ZnPc was detected in the skin, fat or muscle, the maximum concentration occurring at 12 h. In vivo fluorescence reached a maximum intensity approx. 6 h post injection at the mid-chest analysis site and at 12 h in the thigh. The in vivo measurements at two different anatomical sites showed pharmacokinetic behavior that reflects an overall integrated fluorescence originating from several tissue sites.     

PHTHALOCYANINE FLUORESCENCE AT HIGH CONCENTRATION: DIMERS OR REABSORPTION EFFECT?

Abstract— For tetrasulfonated aluminum phthalocyanine (AlPcS₄), dimer formation is characterized in the absorption spectrum by a broadening of the Q-band and the appearance of a new band at the red edge of the spectrum. The high concentrations required to produce dimers, however, often leads to anomalous observations in fluorescence spectroscopy. In the present study, we have examined the photophysical characteristics of two dye systems; AlPcS₄ in a 66% ethanol/water mixture and disulfonated aluminum phthalocyanine in methanol. Using absorption spectroscopy, the formation of dimers is shown to be prevalent only in the case of AlPcS₄. The fluorescence emission spectra in both cases, however, exhibit similar spectral changes with increasing dye concentration. The measured fluorescence decay profiles for both dyes also show similar trends: They are monoexponential, invariant with emission wavelength and have decay times that increase with dye concentration. These distortions are sometimes incorrectly attributed to dimer fluorescence. We find no evidence for the existence of dimer fluorescence and demonstrate that these data can be readily explained, by taking into consideration the effects of reabsorption of fluorescence.     

ABSORPTION AND FLUORESCENCE SPECTROSCOPIC STUDIES ON DIMERIZATION OF CHLOROALUMINUM (III) PHTHALOCYANINE TETRASULFONATE IN AQUEOUS ALCOHOLIC SOLUTIONS

Dimerization of chloroaluminum (III) phthalocyanine tetrasulfonates (AIPCS) has been observed in different aqueous alcoholic solvents at room temperature by absorption and fluorescence spectroscopic methods. Both absorption and fluorescence spectral bands of the dimer are red shifted by ca 550 cm^-1 from the monomer Q bands in the corresponding spectra, suggesting that the interaction energy between the two monomer subunits is very weak. The fluorescence lifetime of the dimer is longer ( ca 9.5 ns) than that of the monomer ( ca 7–8 ns). These spectral behaviors of AIPCS dimer contrast with those of transition-metallaloid phthalocyanine dimers, which usually have a nonfluorescent face-to-face stacking conformation. The dimer fluorescence is interpreted to be due to the fact that the lowest excited singlet state of the dimer is lower in energy than a charge-resonance state, based on the excitoncoupling theory applied to the face-to-face slipping conformation. The dimerization constant determined spectrometrically decreases with an increase of water content in the aqueous alcoholic solution. Propanol and ethanol have been observed to be more effective than methanol in promoting dimerization. These results indicate that a specific interaction of water with AIPCS plays an important role in the inhibition of dimerization of AIPCS.     

PHOTOINDUCED ELECTRON TRANSFER I.INTER-AND INTRAMOLECULAR QUENCHING OF ZINC PHTHALOCYANINE FLUORESCENCE BY ANTHRAQUINONE

Inter-and intramolecular quenching of zinc phthalocyaninefluorescence by anthraquinone has been studied.The diminutions offluorescence quantum yield and life-time are due primarily to electrontransfer.Apparent electron transfer rate constants were calculateddepending on the chain length and solvent polarity.     

PHOTOINDUCED ELECTRON TRANSFER REACTIONS IN PHTHALOCYANINE DISPERSIONS

Abstract— The photochemistry of phthalocyanine particles suspended in a fluid has been investigated. In alcoholic media, these particles were shown to be capable of reducing benzoquinone and oxidizing hydroquinone. In aqueous media the light induced formation of superoxide was detected. This reaction could be substantially enhanced by addition of EDTA. The role of surfactant in the photoproduction of superoxide is associated with the charge acquired by the particle as a result of the surfactant used. These results can be explained in terms of the band model for semiconductors where band bending by surfactant molecules is invoked. Such studies have relevance to photoevents occurring in photosynthesis, photocatalysis and to the types of solar energy conversion systems where a photoactive semiconductor interfaces with an aqueous medium.     

FLUORESCENCE OF TRYPTOPHAN IN KERATIN

The excitation and emission spectra have been determined for the fluorescence from trypto-phan residues in dry keratin. The fluorescence decay was also measured and shown to be a single exponential with a rather long lifetime of 6.9 ns. It is suggested that the emission takes place from a state formed by interaction between the ¹L_a state of the tryptophan residues and neighbouring polar or polarizable groups in the protein. The fluorescence excitation spectrum displays a peak at 290 nm, and its appearance at this position rather than at 280 nm, which is the absorption maximum of tryptophan, is believed to arise from inner filtering by the tyrosine residues in keratin.     

OPTICAL PENETRATION IN HUMAN INTRACRANIAL TUMORS

Abstract— The optical penetration depth has been measured in human intracranial tumors. The penetration depth in highly malignant gliomas is typically twice as large as the penetration depth in the surrounding normal brain tissue. Typical values for different wavelengths are: 1 mm for blue/green, 2.5 mm for red and 6 mm for near infrared. The penetration depth in a metastasis (from lung) is about the same as in the surrounding tissue.     

氧钒酞菁在激光光盘系统中的应用研究

氧钒酞菁在激光光盘系统中的应用研究董长征，沈永嘉，任绳武（华东理工大学精细化工研究所上海２００２３７）袁海俊（中国科学院上海光学精密机械研究所上海２０１８００）关键词：信息储存，激光光盘，功能染料，氧钒酞菁自１９７２年Ｐｈｉｌｉｐｓ公司推出激光光盘（...     

ELECTROSTATIC FORCES IN DYNAMIC FLUORESCENCE QUENCHING

Abstract— The quenching constant for the iodide quenching of the fluorescence of riboflavin phosphate was measured at pH 5 and at pH 8. The value of the constant at the lower pH (where the phosphate group is unionized), k' = 23 M^-1 was independent of the ionic strength, μ. The ratio k"/k'= f(k" the quenching constant at pH 8, where the phosphate group carries a single negative charge) was found to be 0.70 at μ=0.025 and 0.79 at μ= 0.045. The fact that f < 1 is interpreted as due to electrostatic repulsion between the dye molecule, when ionized, and the iodide ion. Due to counter ion screening the effect is less accentuated at higher μ. The quenching constant for riboflavin (which does not contain the ionizable phosphate group) was found to have the same value, 30 M^-1 both at pH 5 and at pH 8, and with no dependence on μ.     

FLUORESCENCE INDUCTION IN ALGAE AND CHLOROPLASTS

Abstract —The first phases of the fluorescence transient elicited by illumination of dark-adapted algae or isolated chloroplasts (biphasic rise φ_***v - φ_I - _P) have previously been shown to be controlled by two quenchers: Q , the primary acceptor of Photosystem 2 interacting with the secondary acceptor pool A ; and R , a non-photochemical quencher which goes into a non-quenching state as A is reduced.
The dependence of the kinetics of φ decay after illumination upon the redox state of A was studied. It is suggested that some of the centres are in a disconnected state, where electron transfer between Q and A is hindered, the amount of such centres being correlated to the reduction state of A . The implications of this hypothesis on the problem of the variation of the Q-A 'equilibrium constant' under different experimental conditions, and on Murata's 'weak light effect' are discussed.
The effect of 3-(3,4 dichlorophenyl)-1,1 dimethylurea (DCMU) on R is shown to depend upon the redox state of A . A DCMU-induced shift of the midpoint potential of R may account for this dependence.
Evidence is given suggesting that the transient reduction of A which occurs in algae during the φ_I-φ_P rise is controlled by an induction process on the acceptor side of Photosystem 1.     

"ENERGY"-DEPENDENT QUENCHING OF CHLOROPHYLL FLUORESCENCE and THERMAL ENERGY DISSIPATION IN INTACT LEAVES DURING INDUCTION OF PHOTOSYNTHESIS

Abstract— Intact leaves, previously adapted to darkness for a prolonged period of time, were suddenly illuminated with a strong, photosynthetically saturating, white light (ca 1500 μmol m⁻² s^_1), resulting in the rapid establishment of a large energy-dependent chlorophyll fluorescence quenching (q_E) as shown by in vivo fluorescence measurements with a pulse amplitude modulation technique. Two different photothermal methods, photoacoustic spectroscopy and photothermal deflection spectroscopy, were used to monitor thermal deactivation of excited pigments during the dark-light transitions. The in vivo photothermal signals measured with both techniques were shown to remain constant during induction of photosynthesis under high light conditions, suggesting that, in contrast to current hypotheses, energy-dependent quenching q_E is not associated with significant changes in thermal dissipation of absorbed light energy in the chloroplasts. When photosynthesis was induced with a low-intensity modulated light, a noticeable decrease in the heat emission yield was observed resulting from the progressive activation of the competing photochemical processes.     

RESONANCE FLUORESCENCE IN CHLOROPHYLL a SOLUTIONS

Abstract— Chlorophyll a in aqueous hexane solutions has two fluorescence peaks, two fluorescence excitation peaks in the blue, and two fluorescence excitation peaks in the red. One of these set of peaks (excitation 2.34_μ^-1 and 1.52_μ-1, fluorescence 1–50_μ^-1) is identical with those found in methanol solutions of chlorophyll a and is assigned to an unaggregated species, while the other set of peaks (excitation 2.44_μ-1 and 1.475_μ^-l, fluorescence 1.48_μ^-l) is assigned to a species aggregated in the presence of water. The spectral shifts occurring in the aggregate follow the selection rules obtained by McRae and Kasha for a linear stack of parallel dye molecules. The sharply increased overlap of absorption and fluorescence leads to resonance fluorescence in the aggregate. The resonance fluorescence is demonstrated experimentally.     

FLUORESCENCE DEPOLARIZATION BY LIGHT-SCATTERING IN TURBID SOLUTIONS

Abstract— The effect of light-scattering on the fluorescence polarization of solutions has been investigated both theoretically and empirically. The criteria by which turbidity depolarization may be recognised are described, and differentiated from those which characterise depolarization through optical rotation or birefringence. These criteria have been used to confirm that the fluorescence depolarization which accompanies actomyosin formation originates in increased light-scattering rather than a real change in relaxation time.     

FLUORESCENCE SPECTRA IN LUNG WITH PORPHYRIN INJECTION

The fluorescence emission spectra from human bronchial mucosa and tumors, before and after injection of dihematoporphyrin ether/ester, have been measured with an optical multichannel analyzer from 500 to 750 nm. Fluorescence was excited with a violet krypton ion laser (average wavelength 410 nm). The autofluorescence spectra decrease monotonically with increasing wavelength except for a small broad peak near 600 nm. The spectra from tumor sites, after injection of the fluorescent porphyrin, exhibit the characteristic fluorescence emission at 630 and 690 nm, added to the autofluorescence spectrum. The spectra from control or nontumor sites are similar but the magnitude of the component due to the injected porphyrin is smaller than at a tumor site. The magnitude ratio of tumor to control site fluorescence depends on concentration of the porphyrin, tumor thickness, and time after injection. Autofluorescence degrades contrast and thus makes very thin tumors difficult to image. Subtraction of the autofluorescence background is desirable.     

OXYGEN QUENCHING OF CHLOROPHYLL FLUORESCENCE IN CHLOROPLASTS

Abstract— Three phases of chlorophyll a fluorescence quenching by O₂ are observed in green plants. The effects of various inhibitors on photosynthetic partial processes in chloroplasts were investigated in attempts to (1) localize the O₂-quenching sites and (2) assess possible physiological significance of O₂-quenching. Our results localize the most sensitive (and presumably functionally important) phase to a site between plastoquinone and the photosystem I acceptor, chlorophyll (P₇₀₀), possibly plastocyanin. It is suggested that PC may transfer electrons to oxygen in addition to P₇₀₀.