Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.     

A DFT study on the vibrational spectroscopy of protoporphyrin IX

Infrared and Raman spectra of protoporphyrin IX were recorded. DFT quantum chemical calculations were performed. Optimised molecular geometry, electric charge distribution, vibrational force constants were computed. The normal coordinate analysis and the scaling of the force constants yielded all the necessary data for the simulation of the infrared and Raman spectra and the potential energy distribution calculations. The result was the interpretation of all vibrational modes of the molecule. Conclusions were drawn from the difficulties arisen during the assignment of the vibrational spectra of such large molecules.     

Subcellular localization pattern of protoporphyrin IX is an important determinant for its photodynamic efficiency of human carcinoma and normal cell lines

Photodynamic therapy (PDT) is a combination of light with a lesion-localizing photosensitizer or its precursor to destroy the lesion tissue. PDT has recently become an established modality for several malignant and non-malignant conditions, but it can be further improved through a better understanding of the determinants affecting its therapeutic efficiency. In the present investigation, protoporphyrin IX (PpIX), an efficient photosensitizer either endogenously induced by 5-aminolevulinic acid (ALA) or exogenously administered, was used to correlate its subcellular localization pattern with photodynamic efficiency of human oesophageal carcinoma (KYSE-450, KYSE-70) and normal (Het-1A) cell lines. By means of fluorescence microscopy ALA-induced PpIX was initially localized in the mitochondria, whereas exogenous PpIX was mainly distributed in cell membranes. At a similar amount of cellular PpIX PDT with ALA was significantly more efficient than photodynamic treatment with exogenous PpIX at killing all the 3 cell lines. Measurements of mitochondrial membrane potential and intracellular ATP content, and electron microscopy showed that the mitochondria were initially targeted by ALA-PDT, consistent with intracellular localization pattern of ALA-induced endogenous PpIX. This indicates that subcellular localization pattern of PpIX is an important determinant for its PDT efficiency in the 3 cell lines. Our finding suggests that future new photosensitizers with mitochondrially localizing properties may be designed for effective PDT.     

Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.     

A comparison of protoporphyrin IX and protoporphyrin IX dimethyl ester as a photosensitizer in poorly differentiated human nasopharyngeal carcinoma cells

Protoporphyrin IX dimethyl ester (PME), a dimethyl esterification of protoporphyrin IX (PpIX), exhibits higher intracellular uptake into NPC/CNE2 cells, a poorly differentiated human nasopharyngeal carcinoma, than does PpIX. Phototoxicity studies reveal PME to be a more potent photosensitizer than is PpIX, at the early and late incubation time points. Correlating phototoxicity with subcellular localization indicates that PME is a more potent photosensitizer when its primary target of photodamage is mitochondria. Also, additional targeting of lysosome enhances phototoxicity.     

Phototoxicity of protoporphyrin IX, diarginine diprotoporphyrinate and N,N-diphenylalanyl protoporphyrin toward human fibroblasts and keratinocytes in vitro: effect of 5-methoxypsoralen

The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N-diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5-methoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet-A (UV-A) irradiation (lambda=365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratinocytes decreases in the order: PPIX > PP(Arg)2 > PP(Phe)2. A synergistic effect of 5-MOP on the phototoxicity of PPIX, PP(Arg)2 and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1-0.5 microM 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)2 and 5-MOP concentrations lower than 0.75 microM. Above this 5-MOP concentration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5-MOP. On the other hand, the intracellular content of PP(Arg)2 is decreased in a concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg)2 phototoxic effect in the presence of 5-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structures of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)2 cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)2 phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg)2 in combination with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.     

Spectroscopic study of protoporphyrin IX zinc(II) encapsulated in sol-gel glass

Previous studies indicated that the organization of native porphyrins and their intentionally designated derivatives in solid substrates is of current interest because of the biological and practical importance of these compounds. In this paper, we report herein for the first time the incorporation of a functionalized diacid, protoporphyrin IX Zn(II) (Zn-pp-IX), successfully in a silica based transparent organic-inorganic hybrid material by choosing proper alkoxy silane containing amino-group via the sol-gel method. The entrapped guest was diagnosed using UV-vis sectrophotometry, emission spectroscopy, and infrared spectrometry; the properties of the encapsulated porphyrin were compared to those of the compound in solution. The results indicate that Zn-pp-IX is well distributed and homogeneously in the glass.     

Gallium(III) protoporphyrin IX]2: a soluble diamagnetic model for malaria pigment

Gallium(III) protoporphyrin IX forms a dimeric propionate-bridged dimer, 2, that is a soluble diamagnetic analogue of hematin anhydride. The single-crystal structure of 2 corresponds to a nondisordered inversion-symmetric dimer similar to malaria pigment but, unlike it, has a six-coordinate metal and an intraporphyrin rather than an interporphyrin hydrogen bond. NMR NOE correlations demonstrate the presence of the propionate linkage in solutions with pyridine. Taken together, this is the first single-crystal X-ray diffraction study of a propionate-linked dimer as found in malaria pigment and the first evidence for its presence in solution.     

Tumor suppressor Fhit protein interacts with protoporphyrin IX in vitro and enhances the response of HeLa cells to photodynamic therapy

Fhit, the product of tumor suppressor fragile histidine triad (FHIT) gene, exhibits antitumor activity of still largely unknown cellular background. However, it is believed that Fhit-Ap(3)A or Fhit-AMP complex might act as a second class messenger in cellular signal transduction pathway involved in cell proliferation and apoptosis. We demonstrate here for the first time that the photosensitizer, protoporphyrin IX (which is a natural precursor of heme) binds to Fhit protein and its mutants in the active site in vitro. Furthermore, PpIX inhibits the enzymatic activity of Fhit. Simultaneously, PpIX shows lower binding capacity to mutant Fhit-H96N of highly reduced hydrolase activity. In cell-based assay PpIX induced HeLa cell death in Fhit and Fhit-H96N-dependent manner which was measured by means of MTT assay. Moreover, HeLa cells stably expressing Fhit or mutant Fhit-H96N were more susceptible to protoporphyrin IX-mediated photodynamic therapy (2J/cm(2)) than parental cells.     

Aminolaevulinic acid-induced protoporphyrin IX pharmacokinetics in central and peripheral arteries of the rat

Photodynamic therapy (PDT) based on the photosensitive protoporphyrin IX (PpIX) may prevent restenosis after transluminal angioplasty. PpIX is synthesized in mitochondria, which differ in number and activity among various tissues. Therefore, we questioned whether the course of PpIX concentration after systemic aminolaevulinic acid (ALA) administration differed among various arteries. ALA was administered intravenously (200 mg/kg) to male Wistar rats (n = 21). At varying time intervals (0, 1, 2, 3, 6, 12 and 24 h) both central and peripheral arteries were isolated and homogenized, and the concentration of the various heme intermediates was determined by a fluorometric extraction method. The maximal PpIX concentration was more than two-fold higher in peripheral arteries (20.49 +/- 3.0 to 24.0 +/- 7.5 pmol/mg protein) than in central arteries (0-9.46 +/- 0.01 pmol/mg protein) (P < 0.004). However, the amount of citrate synthase, reflecting the mitochondrial mass, was lower (0.14-0.61 and 1.87-2.32 U/mg protein, respectively). Apparently, the level of PpIX cannot simply be explained by the mitochondrial content of the arteries. The time interval of maximal PpIX accumulation was similar in peripheral and central arteries (2 h and 27 min vs. 2 h and 8 min) (P = 0.13). Thus, if the efficacy of PDT in vivo is directly related to the tissue concentration of PpIX, more effect can be expected in peripheral arteries than in central arteries.     

An in vivo study of free radicals generated in murine skin by protoporphyrin IX and visible light

Lipids extracted from the skin of C57BL/6J mice injected subcutaneously with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) and exposed to topical protoporphyrin IX (PPIX) and visible light had significantly higher levels of POBN spin adducts compared with dark PPIX exposed or vehicle-treated controls. Computer analysis of the POBN adduct electron paramagnetic (spin) resonance (EPR) spectra indicated that two radical species were present in each extract, one of which was a lipid-derived carbon-centered adduct (1, a(N) = 14.8 G and a(H) = 2.6 G), whereas the other (2, a(N) = 13.8 G and a(H) = 1.8 G) was probably oxygen centered. Adduct 2 was present in greater proportion in lipids extracted from PPIX/light-exposed mice compared with dark or vehicle-treated controls. These findings suggest that PPIX/light generates free radicals in mouse skin, thus providing a radical mechanism for PPIX-induced photosensitivity. Our approach may be useful for the detection of free radicals generated by other skin photosensitizers and may also provide a means for testing putative skin-protecting agents.     

Dihydrocercosporin singlet oxygen production and subcellular localization: a possible defense against cercosporin phototoxicity in Cercospora

Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (1O2)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants. Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity. The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state. As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for 1O2 production in a range of solvents. We found that as a 1O2 photosensitizer, HAC was only moderately effective in organic solvents (phi SO = 0.14-0.18) and very poor in water (phi SO = 0.02-0.04). By contrast, the 1O2 quantum yield of cercosporin itself was unaffected by solvent (phi SO = 0.84-0.97). To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy. This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin. We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular 1O2 production to levels that can be tolerated by the fungus. In addition, we have, for the first time, directly detected 1O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating 1O2 production in vivo and from cercosporin in culture.     

Photodetection with 5-Aminolevulinic acid-induced protoporphyrin IX in the rat abdominal cavity: drug-dose-dependent fluorescence kinetics

In 75% of cases, ovarian carcinoma has already metastasized in the abdominal cavity at the time of diagnosis. For determination of the necessity for a supplementary therapy, in addition to surgical resection, it is important to localize and stage microscopical intraperitoneal metastases of the tumor. Intraperitoneal photodetection of tumor metastases is based on preferential tumor distribution of a fluorescent tumor marker. The time-dependent differences in drug concentration between tumor and normal (T/N) tissues can be used to visualize small tumors. We performed fluorescence measurements on abdominal organs and tumor in the peritoneal cavity of rats. 5-Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was used as the fluorescent marker. Three different drug doses (100, 25 and 5 mg/kg) were used and PpIX fluorescence profiles were followed up to 24 h after intravenous administration. Maximum T/N ratios were found 2-3 h after administration of ALA with all drug doses. A significant T/N tissue contrast was obtained for all abdominal organs tested after administration of 5 mg/kg.     

Synthesis and photochemical properties of a novel iron-sulfur-nitrosyl cluster derivatized with the pendant chromophore protoporphyrin IX

The novel Roussin red-salt ester (PPIX-RSE) with a pendant porphyrin chromophore was prepared and investigated as a precursor for the photochemical generation of nitric oxide. PPIX-RSE has the general formula Fe(2)(NO)(4)[(mu-S,mu-S')P] (where (S,S')P is the bis(2-thiolatoethyl) diester of protoporphyrin IX. The photoexcitation of PPIX-RSE with 436- or 546-nm light in an aerated chloroform solution led to the photodecomposition of the cluster with the respective quantum yields (5.2 +/- 0.7) x 10(-4) and (2.5 +/- 0.5 x 10(-4)) and the concomitant release of NO. PPIX-RSE is a significantly more effective NO generator at longer wavelength excitation than are other Fe(2)(mu-SR)(2)(NO)(4) esters for which R is a simple alkyl group such as CH(3)CH(2)- because of the much higher absorptivity of the pendant PPIX chromophore at these wavelengths and a modestly higher quantum yield. Fluorescence intensity and lifetime data indicate that the photoexcited porphyrin of PPIX-RSE is largely quenched by the energy transfer to the Fe(2)S(2)(NO)(4) cluster's core. However, a small fraction of this emission is not quenched, and it is proposed that PPIX-RSE may exist in solution as two conformers.     

Temporal fluctuations in the SERRS spectra of single iron–protoporphyrin IX molecule

Surface enhanced resonance Raman spectra of Fe–protoporphyrin IX, adsorbed on silver colloidal nanoparticles immobilized onto a polymer-coated glass slide have been investigated at very low concentrations. The spectra exhibit drastic temporal fluctuations on a time scale of seconds in both line frequency and intensity; such a trend suggesting that the single molecule limit is approached. Sequences of spectra have been analyzed in terms of an underlying continuum and of Raman peaks superimposed on this continuum. A statistical analysis of the spectrum intensity has allowed us to put into evidence that main contribution to the intensity fluctuations arises from the continuum. In addition, a high correlation between the total integrated intensity and the intensity detected at different Raman peaks has been revealed. Furthermore, the ratio between the intensity detected in correspondence of different FePP vibrational modes shows a temporal variability likely reflecting the intrinsic dynamics of the molecule. All these findings have been ascribed to a desorption–adsorption mechanism of the molecules at the silver surface.     

Photodegradation pathways and the in vitro phototoxicity of pyrazinamide, a phototoxic antitubercular drug

The phototoxic antitubercular drug pyrazinamide (1) is photolabile under irradiation with UV-A light as well as with a N2 laser (at 337 nm) in aerobic conditions. Irradiation in methanolic and in aqueous solutions of 1 produces four and three photoproducts, respectively. Their formation involves primary alpha-cleavage between the excited carbonyl of the amido group and the aromatic ring followed by hydrogen abstraction and dimerization. Pyrazinamide was able to cause photohemolysis in human erythrocytes and peroxidation of linoleic acid. Inhibition of both processes on addition of reduced glutathione (GSH) or ascorbic acid suggests the involvement of radicals. The absence of inhibition of the photohemolysis and lipid peroxidation processes in the presence of sodium azide (NaN3), or irradiation under argon, and the absence of singlet oxygen during the photolysis confirmed with 2,5-dimethylfuran rules out the possibility of participation of 1O2 in this process. Glutathione depletion was also observed. A radical intermediate was evidenced by thiobarbituric acid that was used as a radical probe, as well as by the dimerization of cysteine. No photohemolysis was detected in presence of the isolated photoproduct. We have also determined the relative efficiencies for the formation of single strand breaks after the irradiation of pBR322 DNA and pyrazinamide, which was also reduced in the presence of GSH.     

A spectroscopic method for simultaneous determination of protoporphyrin IX and hemoglobin in the nerve tissues at intraoperative diagnosis

Russian Journal of General Chemistry - A combined method of spectroscopic analysis of biochemical and structural markers of tumor changes, including blood volume, hemoglobin oxygen saturation,...     

Irradiation of the porphyrin causes unfolding of the protein in the protoporphyrin IX/beta-lactoglobulin noncovalent complex

Porphyrins such as protoporphyrin IX (PPIX) are known to occasionally cause conformational changes in proteins for which they are specific ligands. It has also been established that irradiation of porphyrins noncovalently intercalated between bases or bound to one of the grooves can cause conformational effects on DNA. Conversely, there is no evidence reported in the literature of conformational changes caused by noncovalently bound PPIX to globular proteins for which the porphyrin is not a specific ligand. This study shows that the irradiation of the porphyrin in the PPIX/lactoglobulin noncovalent complex indeed causes a local and limited (approximately 7%) unfolding of the protein near the location of Trp19. This event causes the intrinsic fluorescence spectrum of the protein to shift to the red by 2 nm and the average decay lifetime to lengthen by approximately 0.5 ns. The unfolding of lactoglobulin occurs only at pH >7 because of the increased instability of the protein at alkaline pH. The photoinduced unfolding does not depend on the presence of O2 in solution; therefore, it is not mediated by formation of singlet oxygen and is likely the result of electron transfer between the porphyrin and amino acid residues.     

Intracellular localization of sulfonated meso-tetraphenylporphines in a human carcinoma cell line

The intracellular localization of meso-tetraphenylporphines sulfonated to different degrees (TPPSn), in a human cervix carcinoma cell line (NHIK 3025), was studied by fluorescence microscopy and fluorescence spectroscopy. After an 18 h incubation, TPPS4, TPPS2a and TPPS2o were localized in extranuclear granules. Studies of cells stained with both TPPS4, and acridine orange, which is known to fluoresce red in lysosomes, indicated that these granules were lysosomes. In addition, a fraction of the cellbound TPPS4, TPPS2a and TPPS2o seems to be associated with the plasma membrane. Fluorescence quenching studies of cells doublestained with acridine orange and TPPS4 indicated that TPPS4 is also localized in the nucleus and in the extralysosomal cytoplasm. The intracellular location of TPPS1 differed from that of the other TPPSns studied: In 6 out of 9 experiments fluorescing extranuclear granules were found. A diffuse fluorescence extending from the perinuclear area was also observed.