THE PHOTODEGRADATION OF PORPHYRINS IN CELLS CAN BE USED TO ESTIMATE THE LIFETIME OF SINGLET OXYGEN

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.     

Photoproducts formed from photofrin II in cells

Fluorescence and absorption spectra of light-exposed cells containing the tumour-localizing porphyrin preparation Photofrin II (PII) have been studied. Light exposure results in spectral changes that may be due to a photoinduced modification of the porphyrins without breakage of the porphyrin macrocycle and/or to a photoinduced displacement of the porphyrins in the cells. Photochemical reaction involving breakage of the porphyrin macrocycle also occur as can be seen from the loss of absorbance within the Soret band region during light exposure. Singlet oxygen may be involved in the photodegradation of PII in cells since the process is slowed down on bubbling N2 through the samples and is slightly faster in suspensions in Dulbecco's phosphate buffered saline (PBS) made of D2O compared with suspensions in PBS made of H2O. During light exposure a fluorescent product is formed in the cells with fluorescence excitation and emission characteristics similar to those of the "age pigment" lipofuscin (lambda exc = 350 nm, lambda em = 440 nm).     

CELLULAR DELIVERY AND RETENTION OF PHOTOFRIN II: THE EFFECTS OF INTERACTION WITH HUMAN PLASMA PROTEINS

The absorbance and fluorescence spectra of Photofrin II (PII) in the presence of albumin, globulins and lipoproteins from human plasma show that all of these proteins induce a degree of disaggregation of PII material. In addition, there are substantial rearrangements in the distribution of different fractions contained in PII and their binding to the protein. It is shown that these rearrangements have considerable impact on the uptake of PII by cultured cells and the ensuing retention of the drug in the cells. The information on the contribution of fluorescing and non-fluorescing components of PII in the cells was obtained by measuring first the PII fluorescence in suspensions of live cells, followed by chemical extraction of porphyrin material from the same cells. The interaction of PII with low density lipoproteins resulted in markedly lower levels of PII material retained in the cells, compared to protein-free drug exposure. Somewhat better but still inferior PII retention was observed with high density lipoproteins. The samples with very low density lipoproteins showed increased uptake of PII, but the subsequent retention of the drug was low, so that the remaining amount of the drug was not much different than in protein-free samples. The strongest inhibition of PII uptake was seen with albumin, with ensuing retention of PII not significantly different than in protein-free samples. The best retention of PII was observed with globulins, with approx. 25% higher total drug content retained in the cells after long-term clearance relative to protein-free samples.     

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.     

Time-gated fluorescence spectroscopy of porphyrin derivatives and aluminium phthalocyanine incorporated in vivo in a murine ascitic tumour model.

The effect of systemic administration on drug uptake at cellular level was evaluated using time-gated fluorescence spectroscopy performed on a murine ascitic tumour model. Mice bearing L1210 leukaemia were injected intraperitoneally or intravenously with 25 mg per kg body weight hematoporphyrin derivative (HpD), 12.5 mg per kg body weight photofrin II (PII), 25 or 5 mg per kg body weight disulphonated aluminium phthalocyanine (AlS2Pc). Every 2 h and for up to 22 or 30 h, mice were sacrificed, leukaemic cells extracted from the peritoneum, washed, and resuspended in buffer for fluorescence measurements. HpD and PII emission spectra were almost identical 12 h after intraperitoneal injection with main peaks at 630 nm and no appreciable changes afterwards. In the first 12 h, the PII fluorescence spectrum was constant, while in the case of HpD a shoulder at 615 nm was detectable. Similar fluorescence behaviour was observed after intravenous administration of porphyrin derivatives. These results seem to confirm that the tumour localizing fraction is the part actually retained by the cells. The AlS2Pc spectrum peaked at 685 nm and did not change in any of our experiments. AlS2Pc is incorporated more rapidly with respect to porphyrins, as was clearly observed in the case of intravenous administration, where the AlS2Pc fluorescence was readily detectable after 2 h, whereas the PII emission became apparent only after 4-6 h.     

Aggregation and solution behavior of 5-(1-(4-carboxybutyl) pyridinum-4-yl) 10,15,20-tris (1-methylpyridinium-4-yl) porphyrin: A resonance light scattering, fluorescence and absorption spectroscopic study

Aggregation behavior of water soluble porphyrins, 5-(1-(4-carboxybutyl) pyridinum-4-yl) 10,15,20-tris (1-methylpyridinium-4-yl) porphyrin (5-CBPyP) in the presence of various concentrations of calf thymus DNA (ct-DNA) and sodium chloride were studied in comparison with meso-tetrakis (4-N-methyl pyridinum) porphyrin (TMPyP), by optical absorption, fluorescence and resonance light scattering (RLS) spectroscopies. Both porphyrins obey Beer’s law in extended range of concentration. Optical absorption and RLS measurements demonstrated nonaggregation for both porphyrins under increasing concentration of ct-DNA and NaCl. However, in comparison, 5-CBPyP had less tendency for aggregation that may be taken as an advantage for its probable application in photodynamic therapy of cancer. The trend of changes in absorption spectra of both porphyrins in the presence of ct-DNA indicates the homogeneous intercalation binding mode. The values of (2.81 ± 0.28) × 10⁶ M^?1 and (0.95 ± 0.09) × 10⁶ M^?1 were obtained for apparent binding constant of TMPyP and 5-CBPyP from analysis of optical absorption data, respectively. This indicates the less affinity of 5-CBPyP to ct-DNA in comparison with TMPyP. The binding of both porphyrins to ct-DNA quenches fluorescence emission of Ethidium bromide (EB) that is bound to ct-DNA. The quenching process obeys linear Stern-Volmer relationship indicating the displacement of EB from its binding sites by these porphyrins. The results of this technique also represent the intercalation mode of binding for both porphyrins and higher binding affinity of TMPyP compared with 5-CBPyP.     

Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light

Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.     

The photosensitizing effect of the photoproduct of protoporphyrin IX

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm² light at 635 nm, the photoproduct — the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.     

Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers.

The variations of fluorescence during light exposure of the cationic sensitizers methylene blue (MB) and meso-tetra(4N-methylpyridyl)porphyrin (T4MPyP) as well as the anionic meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) were measured at different intracellular sites using video-intensified microscopy in combination with microspectrofluorometry. Before light exposure the sensitizers were localized in distinct parts of the cytoplasm, especially in fluorescent organelles. During irradiation a drastic fluorescence formation and increase in the cytoplasm and nucleus, which was most pronounced in the nucleoli, could be observed for the cationic sensitizers as well as TPPS4. In the case of MB the increase in fluorescence was concomitant with a spectral shift in the emission spectra. For TPPS4 and T4MPyP the formation of a second species with a Soret band shifted towards longer wavelengths was observed and correlated with the fluorescence increase in the nucleoli. Cell deformations also took place.     

PHOTOSENSITIZERS IN ORGANIZED MEDIA: SINGLET OXYGEN PRODUCTION AND SPECTRAL PROPERTIES

Abstract— Measurement of singlet oxygen production by porphyrin-type photosensitizers in simple buffer solutions show that TPPS is more efficient than PII (Photofrin II) or than hematoporphyrin. This behavior was observed using three independent analytical detection methods for ¹O₂: RNO bleaching, tryptophan degradation, and oxygen consumption. Similar results were obtained when irradiating with a classical light source or with a pulsed dye laser. Addition of EPC liposomes to the buffer solution caused a decrease of efficiency for TPPS and an increase for PII. Addition of BSA shows the same relative pattern: a small increase of efficiency for TPPS and a tenfold increase for PII. These changes can be ascribed to specific interactions between the sensitizer molecules and the organized medium. Since changes in the fluorescence properties are also due to interactions with the medium, we monitored the emission of the sensitizers as a function of the environment. The fluorescence peak positions (at 648, 705 nm for TPPS and at 615, 677 nm for PII) were all red shifted. The intensities show an increase, particularly for PII in liposomes, due to a marked change in the microviscosity.     

Competition between photobleaching and fluorescence increase of photosensitizing porphyrins and tetrasulphonated chloroaluminiumphthalocyanine

The time-dependent fluorescence changes of photosensitizing porphyrins and tetrasulphonated chloroaluminiumphthalocyanine (A1C1SPc) were measured at different intracellular sites using video-enhanced microscopy and image processing. To obtain variations in intracellular fluorescence intensity, different radiant exposures of a Kr+ laser-pumped dye laser were delivered via a 600 microns plastic-clad silica fibre connected to the microscope. During irradiation, competition between photobleaching and fluorescence increase of the different dyes was observed. The porphyrins normally showed photobleaching, which was dependent on the sensitizer and its specific accumulation within the cell. Photobleaching was less pronounced for hydrophilic uroporphyrin than for more hydrophobic dyes. In contrast with an almost exponential decrease in porphyrin fluorescence with increasing light dose, the fluorescence intensity of A1C1SPc significantly increased at the beginning of irradiation, and could be correlated with intracellular deaggregation.     

Changes in the structure of chlorophyll-protein complexes and excitation energy transfer during photoinhibitory treatment of isolated photosystem I submembrane particles

The activity of light-induced oxygen consumption, absorption spectra, low temperature (77 K) chlorophyll fluorescence emission and excitation spectra were studied in suspensions of photosystem (PS) I submembrane particles illuminated by 2000 microE m(-2) s(-1) strong white light (WL) at 4 degrees C. A significant stimulation of oxygen uptake was observed during the first 1-4 h of photoinhibitory treatment, which rapidly decreased during further light exposure. Chlorophyll (Chl) content gradually declined during the exposure of isolated PSI particles to strong light. In addition to the Chl photobleaching, pronounced changes were found in Chl absorption and fluorescence spectra. The position of the major peak in the red part of the absorption spectrum shifted from 680 nm towards shorter wavelengths in the course of strong light exposure. A 6-nm blue shift of that peak was observed after 5-h illumination. Even more pronounced changes were found in the characteristics of Chl fluorescence. The magnitude of the dominating long-wavelength emission band at 736 nm located in untreated particles was five times reduced after 2-h exposure, whereas the loss in absolute Chl contents did not exceed 10% of its initial value. The major peak in low-temperature Chl fluorescence emission spectra shifted from 736 to 721 nm after 6-h WL treatment. Individual Chl-protein complexes differed in the response of their absorption spectra to strong WL. Unlike light-harvesting complexes (LHC), LHCI-680 and LHC-730, which did not exhibit changes in the major peak position, its maximum was shifted from 678 to 671 nm in CPIa complex after PSI submembrane particles were irradiated with strong light for 6 h. The results demonstrated that excitation energy transfer represents the stage of photosynthetic utilization of absorbed quanta which is most sensitive to strong light in isolated PSI particles.     

Investigation on the Photodissociation of Oxygen from Oxymyoglobin by Fluorescence Spectroscopy

Photodissociation of oxygen from oxymyoglobin(oxyMb) was investigated by means of fluorescence spectroscopy. One of the most important findings of the photodissociation of oxyMb was the discovery of two processes which were affected by excitation intensity, temperature, solvent viscosity, and excitation wavelength. Process I(PI) corresponded to oxygen escaping from the binding site at ferrous heme iron atom within the porphyrin ring into the heme pocket, whereas process II(PII) was ascribed to oxygen escaping from the heme pocket into the solvent. To elucidate this interesting phenomenon, we proposed a model that oxygen encountered two barriers on its way from the binding site at the ferrous heme iron to the solvent. Reversibility and wavelength sensitivity of the photodissociation were also observed.     

Multi-spectroscopic Investigations of Aspirin and Colchicine Interactions with Human Hemoglobin: Binary and Ternary Systems

The interactions of colchicine (COL) and aspirin (ASA) with human hemoglobin (HB) was studied by fluorescence, UV/vis absorption, resonance light scattering, synchronous fluorescence and circular dichroism (CD) spectroscopic techniques under physiological conditions. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were determined by the fluorescence quenching technique at different temperatures. The results proved that the mechanism of fluorescence quenching of HB by COL and ASA is due to formation of HB–drug complexes in the binary and ternary systems. The distance between the acceptor drugs and HB was estimated by Förster’s equation on the basis of fluorescence energy transfer. In addition, according to the synchronous fluorescence spectra of HB, the results showed that the fluorescence quenching of HB originated solely from the tryptophan residues and indicated a conformational change for HB caused by addition of the drugs. Far-UV CD spectra of HB were recorded before and after the addition of ASA and COL both as binary and ternary systems. An increase in intensity of the positive CD peak of HB was observed in the presence of these drugs. The results were interpreted as excited state interactions between the aromatic residues of the HB binding sites and the drugs bound to them.     

Belt-shaped π-systems: relating geometry to electronic structure in a six-porphyrin nanoring

Linear π-conjugated oligomers have been widely investigated, but the behavior of the corresponding cyclic oligomers is poorly understood, despite the recent synthesis of π-conjugated macrocycles such as [n]cycloparaphenylenes and cyclo[n]thiophenes. Here we present an efficient template-directed synthesis of a π-conjugated butadiyne-linked cyclic porphyrin hexamer directly from the monomer. Small-angle X-ray scattering data show that this nanoring is shape-persistent in solution, even without its template, whereas the linear porphyrin hexamer is relatively flexible. The crystal structure of the nanoring-template complex shows that most of the strain is localized in the acetylenes; the porphyrin units are slightly curved, but the zinc coordination sphere is undistorted. The electrochemistry, absorption, and fluorescence spectra indicate that the HOMO-LUMO gap of the nanoring is less than that of the linear hexamer and less than that of the corresponding polymer. The nanoring exhibits six one-electron reductions and six one-electron oxidations, most of which are well resolved. Ultrafast fluorescence anisotropy measurements show that absorption of light generates an excited state that is delocalized over the whole π-system within a time of less than 0.5 ps. The fluorescence spectrum is amazingly structured and red-shifted. A similar, but less dramatic, red-shift has been reported in the fluorescence spectra of cycloparaphenylenes and was attributed to a high exciton binding energy; however the exciton binding energy of the porphyrin nanoring is similar to those of linear oligomers. Quantum-chemical excited state calculations show that the fluorescence spectrum of the nanoring can be fully explained in terms of vibronic Herzberg-Teller (HT) intensity borrowing.     

Study on spectroscopic characterization of Cu porphyrin/Co porphyrin and their interactions with ctDNA

The luminescence behaviors of Cu(II) meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin (Cu(II)TMPyP) and Co(II) meso-tetrakis (4-N-methyl-pyridiniumyl) porphyrin (Co(II)TMPyP) are investigated, and their interactions with calf thymus DNA (ctDNA) are studied by UV-vis, fluorescence and resonance light scattering (RLS) and based on the changes of UV-vis spectra, fluorescence and RLS spectra, the intrinsic binding constants of Cu(II)TMPyP/Co(II)TMPyP with ctDNA are obtained in the case of phosphate buffer solution (pH 7.2), respectively. According to the experimental results, it can be inferred that the interaction model of Cu(II)TMPyP with ctDNA is intercalative binding, while Co(II)TMPyP is the long-range assembly on the molecular surface of ctDNA.     

四苯基卟啉镁的合成、表征及光化学性质

提出一种以乙酸镁和乙酸钠为原料合成四苯基卟啉镁(MgTPP)的新方法, 合成样品以柱层析法进行分离纯化. 分离产物经UV-Vis、1H-NMR、MALDI-TOF-MS(基质辅助激光解吸电离飞行时间质谱)等技术表征, 确定为MgTPP. UV-Vis光谱分析结果表明, 四苯基卟啉镁的Soret 吸收带为424 nm, Q 吸收带为563 nm 和602 nm. 此外, 光照对MgTPP的二氯甲烷溶液光谱性质的影响结果表明, 经光照射后MgTPP的UV-Vis光谱的Soret吸收带吸收强度明显降低, 同时, 经550 nm的光激发产生的荧光有明显的猝灭. 对光照后的MgTPP样品进行MALDI-TOF-MS分析, 发现有新的质核比(m/z)出现, 其为668, 这一结果表明, 在光照条件下, MgTPP分子可能与氧分子发生光化学作用, 形成MgTPP与氧的复合物MgTPP-O2.     

Photophysical properties of carborane-containing derivatives of 5,10,15,20-tetra(p-aminophenyl)porphyrin

Absorption, fluorescence emission, fluorescence excitation spectra and fluorescence decay kinetics of carborane derivatives of 5,10,15,20-tetra(p-aminophenyl)porphyrin have been investigated. Carborane derivatives are prepared by acylation of the amino groups of 5,10,15,20-tetra(p-aminophenyl)porphyrin by 9-carboranyl acetyl chloride. From the analysis of the absorption and fluorescence spectra, it is concluded that covalent linking of carborane molecules to the tetraphenylporphyrin molecule significantly changes the self-conjugated pi-system of the porphyrin macrocycle: positions of maxima of absorption and fluorescence spectra shift to the red region by 3-8 nm; the halfwidths of these bands are broadened by 2.5-5.0 nm; the relative intensity of the bands I-IV also changes. The fluorescence decay kinetics of the carborane derivatives are biexponential. According to the experimental data and model simulation, it is concluded that the intramolecular electron transfer proceeds from the porphyrin excited part of the molecule to carboranyls with a rate constant of 415 ps(-1) and efficiency of 0.16-0.8. Recombination of separated charges occurs within 1.4 ns.     

Meso-四（4-（N-咔唑）丁烷氧苯基）卟啉稀土金属配合物的合成及光电性质

本文设计合成了未见文献报道的meso—N（4-（N-咔唑）丁烷氧苯基）卟啉（1）及其稀土金属配合物（Ln=Sm（2）,Eu（3）,Tb（4）,Dy（5））．通过核磁共振氢谱、紫外．可见吸收光谱、元素分析和红外光谱对稀土金属卟啉配合物进行了表征,并研究了卟啉样品的荧光性质和表面光电压性质．结果表明,受无辐射跃迁等因素的影响,卟啉1～5的荧光强度和荧光量子效率依次减小．在无外加电场的情况下,稀土金属配合物2～5的表面光电压强度变化与荧光强度变化呈现相反的规律,充分表明荧光发射与表面光电压的相互竞争过程．不同性质的电场对金属卟啉配合物的电荷分离影响显著,在正电场和无外加电场情况下,配合物2～5的表面光电压信号强度依次增加．     

新型尾式组氨酸卟啉的合成及性质

设计并合成了新型尾式氨基酸卟啉5-(Trt-组氨酸酰胺基苯基)-10,15,20-三苯基卟啉(Trt-His-NH2-TPP,1)及其金属卟啉配合物(Zn,Co,Fe,Mn,分别标记为2～5).通过元素分析、核磁共振氨谱、质谱、紫外-可见光谱和红外光谱等对化合物进行了表征,研究了它们的荧光性质,并通过理论模拟研究了其最...