CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY

Abstract
The clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5–6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero-and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N -methylated porphyrins show clearance times in the vicinity of 15–22 h. Monoaspartyl chlorin e₆ shows the longest clearance time of all porphyrin derivatives measured (30.3 h).     

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.     

PROBING THE STRUCTURE OF HPD BY FLUORESCENCE SPECTROSCOPY

Fluorescence emission spectra indicate that oligomers containing both hematoporphyrin and its dehydration products (vinyl porphyrins) comprise the tumor-localizing fraction of HPD. In the relatively polar solvent methanol, the vinyl porphyrins exhibit reduced fluorescence yields while the hematoporphyrin residues are relatively resistant to fluorescence quenching by Fe+3. In the less polar solvent tetrahydrofuran, fluorescence from oligomeric vinyl porphyrins was enhanced, and Fe+3-induced quenching of oligomeric hematoporphyrin promoted. These, together with other studies in biological systems, suggest a substantial degree of interaction among the porphyrin units contained in these oligomers, as a function of the polarity of the environment.     

Hematoporphyrin derivatives: an oligomeric composition study

The oligomeric composition of HpD, Photofrin II and other hematoporphyrin derivatives useful for the diagnosis and therapy of tumors has been studied. Gel chromatographic procedures were used that excluded porphyrin aggregation. Photofrin and hematoporphyrin derivatives were shown to contain different quantities of monomer, dimer and other oligomeric porphyrins.     

In vitro UPTAKE OF DICARBOXYLIC PORPHYRINS BY HUMAN ATHEROMA. KINETIC and ANALYTICAL STUDIES

Human atheromatous aorta segments as well as presumably disease-free control aorta were obtained at autopsy. They were incubated with solutions of various purified dicarboxylic porphyrins including hematoporphyrin (HP) and hydroxyethylvinyldeuteroporphyrin (HVD), and with solutions of Photofrin. Selective labelling of the atheroma was shown by macroscopic and microscopic observations of the characteristic porphyrin fluorescence associated with the atheromatous plaques. The time dependence of the uptake, monitored by absorption spectrophotometry or by high performance liquid chromatography, was inferred from the disappearance of the porphyrins in the incubation medium. Significant binding was observed in the absence of albumin or serum proteins. The uptake of HP was less than that of the more hydrophobic compounds HVD or Photofrin when these porphyrins were used alone. The presence of albumin or serum drastically reduces atheroma labelling. Some competition between HP and HVD for binding sites is also seen. The present results do indicate that hydrophobic porphyrins have an intrinsic affinity for atheroma and that they can be taken up through passive processes. Taking into account previous data on animal models (Photochem. Photobiol. (1989), 731-737), it appears however that, in vivo, interactions with proteins and pharmacokinetics will primarily determine plaque labelling.     

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.     

QUANTITATIVE in vivo MEASUREMENT OF THE FLUORESCENT COMPONENTS OF PHOTOFRIN II

Abstract Photofrin II which contains the most efficient components of hematoporphyrin derivative with regard to photodynamic therapy of cancer, was measured fluorometrically in tumor and tumor-free tissues in vivo over a period up to 8 days. Using time-resolving (nanosecond and picosecond) microscopic techniques, the fluorescence of different components was quantitated and attributed to monomelic, dimeric, and possibly aggregated porphyrin species. The long-lasting retention of the porphyrins in live tissues was in contrast to the rapid removal from cultured cells. This might be due to monomerization or dimerization of non-fluorescent aggregates. Tumor-selective accumulation was found to be similar for two different (probably monomeric and dimeric) components. This indicates that the integral fluorescence of these components may also correlate with the distribution of the main photosensitizing species.     

PICOSECOND FLUORESCENCE OF R3230AC MAMMARY CARCINOMA MITOCHONDRIA AFTER TREATMENT WITH HEMATOPORPHYRIN DERIVATIVE and PHOTOFRIN® II in vivo

Hematoporphyrin derivative (HPD) and other porphyrin samples were excited by 20-ps 532-nm laser pulses. Fluorescence was detected using a low-jitter streak camera. Data were fitted to a sum of exponential decay times on the order of picoseconds. Fluorescence of porphyrins in aqueous solution show various behaviors depending on the hydrophobicity of the porphyrins. The most hydrophilic porphyrins show long decays only (greater than 500 ps). Porphyrins intermediate in hydrophobicity have intensity-dependent fast decays. The most hydrophobic have fast decays (less than 20 ps). Picosecond fluorescences of mitochondria prepared from rat tumors treated in vivo with HPD or Photofrin II show an increase in the ratio of fast to slow decays when compared to the injected porphyrins. These results are consistent with the concentration of the more hydrophobic porphyrins in mitochondria in photosensitization treatment. Thus picosecond fluorescence studies of porphyrins may provide a means to obtain photoproperties which differentiate between effective and ineffective in vivo photosensitizers.     

Directly meso-meso linked porphyrin rings: synthesis, characterization, and efficient excitation energy hopping

Directly meso-meso linked porphyrin rings CZ4, CZ6, and CZ8 that respectively comprise four, six, and eight porphyrins have been synthesized in a stepwise manner from a 5,10-diaryl zinc(II) porphyrin building block. Symmetric cyclic structures have been indicated by their very simple (1)H NMR spectra that exhibit only a single set of porphyrin and their absorption spectra that display a characteristic broad nonsplit Soret band around 460 nm. Energy minimized structures calculated at the B3LYP/6-31G* level indicate that a dihedral angle between neighboring porphyrins decreases in order of CZ6 > CZ8 > CZ4, which is consistent with the (1)H NMR data. Photophysical properties of these molecules have been examined by the steady-state absorption, fluorescence, fluorescence lifetime, fluorescence anisotropy decay, and transient absorption measurements. Both the pump-power dependence on the femtosecond transient absorption and the transient absorption anisotropy decay profiles are directly related with the excitation energy migration processes within the porphyrin rings, where the exciton-exciton annihilation time and the polarization anisotropy rise time are well described in terms of the Forster-type incoherent energy hopping model. Consequently, the excitation energy hopping rates have been estimated for CZ4 (119 +/- 2 fs)(-)(1), CZ6 (342 +/- 59 fs)(-)(1), and CZ8 (236 +/- 31 fs)(-)(1), which reflect the magnitude of the electronic coupling between the neighboring porphyrins. Overall, these porphyrin rings serve as a well-defined wheel-shaped light harvesting antenna model in light of very efficient excitation energy hopping along the ring.     

Pharmacokinetics of Photogem using fluorescence monitoring in Wistar rats

In this study we investigated the pharmacokinetics of a hematoporphyrin derivative (Photogem) in Wistar rats using the fluorescence spectroscopy to evaluate the drug distribution in liver, kidney and skin tissues. The detection system is composed of a 532 nm exciting laser, a Y-type catheter for light delivery and collection, a monochromator and a computer for data acquisition. The analysis of the fluorescence spectra was based on the intensity of porphyrin emission bands from specific tissues of the investigated organ. A simple transport model is proposed to determine the accumulation and elimination times for each type of investigated tissue. The obtained results show the viability of the fluorescence spectroscopic technique for the drug concentration monitoring in different target tissues and related pharmacokinetics. These effects should be considered before any in vivo study of Photodynamic Therapy using Photogem.     

Synthesis, characterization, and photoinduced electron transfer in functionalized single wall carbon nanohorns

Single-wall carbon nanohorns (SWNHs) are a new class of material that is closely related to single-wall carbon nanotubes. Here, we describe the synthesis and characterization of a series of SWNHs functionalized with ethylene glycol chains and porphyrins. Functionalization of carbon nanohorns has been achieved using two different synthetic protocols: (1) direct attack of a free amino group on the nanohorn sidewalls (nucleophilic addition) and (2) amidation reaction of the carboxylic functions in oxidized nanohorns. The nanohorn derivatives have been characterized by a combination of several techniques, and the electronic properties of the porphyrin/nanohorn assemblies (SWNH/H2P) have been investigated by electrochemistry, spectroelectrochemistry, and a series of steady-state and time-resolved spectroscopy. The cyclic voltammetry curve of nanohorn/porphyrin conjugate 6 showed a continuum of faradic and pseudocapacitive behavior, which is associated with multiple-electron transfers to and from the SWNHs. Superimposed on such a pseudocapacitive current, the curve also displays three discrete reduction peaks at -2.26, -2.57, and -2.84 V and an oxidation peak at 1.12 V (all attributed to the porphyrin moiety). Steady-state and time-resolved fluorescence demonstrated a quenching of the fluorescence of the porphyrin in SWNH/H2P conjugates 5 and 6 compared to the reference free base porphyrin. Transient absorption spectra permitted the electron-transfer process between the porphyrins and the carbon nanostructures to be highlighted.     

THE PHOTOCHEMICAL YIELD OF SINGLET OXYGEN FROM PORPHYRINS IN DIFFERENT STATES OF AGGREGATION

Abstract— Aqueous solutions of hematoporphyrin and hematoporphyrin derivatives were exposed to light. When present in such solutions tryptophan is degraded by a singlet oxygen mechanism. This is true for excitation at 396 nm, where porphyrin monomers have their absorption maximum, as well as for excitation at 360 nm, where porphyrin aggregates seem to absorb strongly. The quantum yield of singlet oxygen production is similar within 25% for excitation at 396 and 360 nm while the fluorescence quantum yield is more than a factor 2 lower for excitation at 360 nm than for excitation at 396 nm. Photoexcitation of the clinically used hematopotophyrin derivatives photofrin I and photofrin II produces singlet oxygen with significantly smaller yields than photoexcitation of hematoporphyrin. Thus, the aggregates present in solutions of photofrin I and photofrin II are of a different nature than those present in aqueous solutions of hematoporphyrin.     

PHOTOSENSITIZATION BY DIPORPHYRINS JOINED VIA METHYLENE BRIDGES

Abstract— Photodynamic and biophysical properties of three porphyrin dimers joined by methylene bridges were examined. Fluorescence emission spectra and fluorescence lifetimes of the methylene-linked dimers were similar to values obtained with porphyrin monomers. Singlet oxygen quantum yields were not significantly different when the three diporphyrins were compared. The diporphyrins were short-acting tumor photosensitizers in vivo, and were rapidly cleared from plasma. Of the 3 diporphyrins examined, one was essentially ineffective as a sensitizer in vivo. This could not have been predicted from in vitro studies which indicated photodamage to membrane and mitochondrial loci. The methyiene-linked diporphyrins were hydrophilic dyes (water/octanol distribution ratio =120–200) and bound mainly to plasma high-density lipoprotein. In contrast, the more hydrophobic diporphyrin ester/ether fraction from HPD was a long-persisting photosensitizer in vivo. Compared with hematoporphyrin, this hematoporphyrin derivative (HPD) fraction demonstrated a red-shift in fluorescence emission and a shortened fluorescence lifetime. These comparisons suggest that ring-ring interactions occur in the ester/ether-linked diporphyrins from HPD, but not in the methyiene-linked diporphyrins.     

The 5-aminolevulinic acid-induced porphyrin biosynthesis in benign and malignant cells of the skin.

In fluorescence diagnosis and photodynamic therapy of neoplastic tissues 5-aminolevulinic acid is used to synthesize endogenous porphyrins as photosensitizers. The efficacy of neoplastic tissues to fluorescence diagnosis and photodynamic therapy is thought to be dependent on the total level of intralesional formed porphyrins. The available profiles of porphyrin metabolites in normal and in neoplastic cell lines after administration of 5-aminolevulinic acid vary considerably. Thus, this is the first in-vitro study which compares the porphyrin biosynthesis in normal skin cells (HaCaT, fibroblasts) with melanoma cells (Bro, SKMel-23, SKMel-28). After incubation with 1 mM 5-aminolevulinic acid, kinetics of porphyrin levels and metabolites were determined in the cells and the corresponding supernatants. Exogenous 5-aminolevulinic acid induced porphyrin formation in all cells with maximum values after an incubation period of 16-36 h. Increase of porphyrin levels varied from 10- to 80-fold (SKMel-28>HaCaT>fibroblasts>SKMel-23>Bro) with minimum 1.5 times higher levels of porphyrins in the supernatants than in the cells. In cells and supernatants protoporphyrin and coproporphyrin were the predominantly formed porphyrin metabolites. Metastatic melanoma cells (SKMel-23, SKMel-28) accumulated much higher porphyrin levels than primary melanoma cells (Bro). In conclusion, by optimizing the treatment modalities, especially the light source, topical photodynamic therapy (PDT) could become a treatment alternative of melanoma metastases in progressive disease.     

PHOTOPHYSICAL AND PHOTOBIOLOGICAL PROPERTIES OF DIPORPHYRIN ETHERS

Spectral properties of several diporphyrin ethers were assessed in different solvents and after accumulation by leukemia L1210 cells in vitro. To facilitate studies in a variety of solvents, both tetramethylesters of the diporphyrin ethers and free acids were employed. For comparison, studies on the corresponding porphyrin monomers were also carried out. The joining of two porphyrins by an ether linkage had several consequences. We observed a blue shift in the Soret band of the ethers, but not of the corresponding simple porphyrins, in protic solvents. This phenomenon is likely related to ether aggregation under conditions which promote H-bonding. The presence of an ether linkage was associated with enhanced fluorescence at 630-640 nm and decreased fluorescence lifetimes and yields, especially in protic solvents. The ether linkage was unaffected by intracellular enzymes, but porphyrin esters were readily hydrolyzed upon accumulation by L1210 cells. The joining of two hematoporphyrin molecules by an ether linkage promoted dye accumulation by L1210 cells. In contrast, accumulation of mesoporphyrin and protoporphyrin was thereby retarded.     

Interactions of dicarboxylic porphyrins with membranes in relation to their ionization state

The interactions of dicarboxylic porphyrins with membrane systems are discussed with particular emphasis on the effect of the charge of the porphyrin and the nature of the side-chains. The incorporation of hematoporphyrin or related dicarboxylic porphyrins within small unilamellar vesicles as membrane models is favored by a decrease of the pH in the range of physiological pH values. This effect might play an important role in the retention of porphyrins by tumors, which are more acidic than normal tissues. Kinetics studies also show that the partition of the porphyrin between the lipidic bilayer and the aqueous phase is governed by its release rate rather than by its incorporation rate.     

DISTRIBUTION OF PORPHYRINS IN THE VARIOUS COMPARTMENTS OF UNILAMELLAR LIPOSOMES OF DIPALMITOYL-PHOSPHATIDYLCHOLINE AS PROBED BY FLUORESCENCE SPECTROSCOPY

Abstract— The spectroscopic properties of hematoporphyrin, hematoporphyrin-dimethyl ester, uroporphyrin and uroporphyrinoctamethyl ester, incorporated into unilamellar liposomes of dipalmitoylphos-phatidylcholine, have been studied with the aim to assess the distribution of porphyrins within the various liposomal compartments.
The results obtained indicate that the highly hydrosoluble uroporphyrin is partitioned in the endoliposomal aqueous pool while its octamethylester is homogeneously distributed in the inner lipid monolayer. Hematoporphyrin and its dimethylester show an heterogeneous distribution within the phospholipid bilayer. At T = 25°C these porphyrins are preferentially located in the outer phospholipid monolayer.
Detailed studies on hematoporphyrin indicate that the distribution between the inner and outer phospholipid monolayer is a function of temperature and liposome dimensions. In particular, the increase of temperature above the critical temperature for the liquid-gel phase transition of the liposomes causes a partial shift of the porphyrin molecules toward the inner phospholipid monolayer. Moreover, the increase of liposome dimensions leads to a greater accessibility of porphyrin to the external medium.     

UPTAKE and PHOTODYNAMIC EFFICIENCY OF HEM ATOPORPH YRIN, HYDROXYETHYLVINYLDEUTEROPORPHYRIN and HEMATOPORPHYRIN DERIVATIVE (PHOTOFRIN II®): A STUDY WITH ISOLATED MITOCHONDRIA

The uptake of Photofrin II (PFII), hematoporphyrin (Hp) and hydroxyethylvinyldeuteroporphyrin (HVD) by isolated mitochondria was studied using the high performance liquid chromatography (HPLC) technique. The various PFII components show a high affinity for mitochondria. At 5.75 micrograms/ml PFII, their ratio of incorporation was found to be very similar, except for Hp which is about two times less incorporated. These results were reproduced with pure Hp and pure HVD. The uptake of Hp and HVD increases with concentration but, while that of Hp reaches a plateau, the uptake of HVD continues to increase. At a high porphyrin concentration (approximately 10(-5) M), the loss of respiratory control is obtained with the same light dose for Hp and PFII. Taking into account the uptake and the known photophysical parameters of the various porphyrins, the photodynamic efficiency of HVD seems equivalent to that of Hp. The present results and known data on cell photoinactivation suggest that the activity of these porphyrins is mainly dependent on their incorporation.     

Fluorescence and absorption spectroscopic studies on the interaction of porphyrins with snake gourd (Trichosanthes anguina) seed lectin

The interaction of several free-base porphyrins and their corresponding copper(II) and zinc(II) derivatives with the galactose-specific lectin from snake gourd (Trichosanthes anguina) seeds has been investigated by absorption and fluorescence spectroscopic techniques. The lectin dimer contains two apparently equivalent binding sites for the porphyrins. Association constants obtained for the interaction of various porphyrins with the lectin are in the range 1.7 x 10(4)-6.2 x 10(5) M(-1), with the metalloporphyrins being seen to have higher affinity for the lectin compared with the free-base analogues. Both positively charged and negatively charged porphyrins bind to snake gourd seed lectin (SGSL) with comparable affinities, suggesting that binding occurs primarily via hydrophobic interactions. Further, binding of porphyrins is found to be largely unaffected by the presence of the sugar ligand, lactose, indicating that the binding sites for the carbohydrate and porphyrin are different. This study thus suggests that the lectin may serve as a receptor for some endogenous non-carbohydrate, hydrophobic ligand in vivo, in addition to the saccharide ligands. It also opens up the possibility of employing the T. anguina lectin in applications such as photodynamic therapy, which involve the use of porphyrins.     

Amino Acid–Porphyrin Conjugates: Synthesis and Study of their Photophysical and Metal Ion Recognition Properties

Synthesis, photophysical and metal ion recognition properties of a series of amino acid‐linked free‐base and Zn‐porphyrin derivatives (5–9) are reported. These porphyrin derivatives showed favorable photophysical properties including high molar extinction coefficients (>1 × 10⁵ m ^?1 cm^?1 for the Soret band), quantum yields of triplet excited states (63–94%) and singlet oxygen generation efficiencies (59–91%). Particularly, the Zn‐porphyrin derivatives, 6 and 9 showed higher molar extinction coefficients, decreased fluorescence quantum yields, and higher triplet and singlet oxygen quantum yields compared to the corresponding free‐base porphyrin derivatives. Further, the study of their interactions with various metal ions indicated that the proline‐conjugated Zn‐porphyrins (6 and 9) showed high selectivity toward Cu²⁺ ions and signaled the recognition through changes in fluorescence intensity. Our results provide insights on the role of nature of amino acid and metallation in the design of the porphyrin systems for application as probes and sensitizers.