Fluorescence localization and kinetics of mTHPC and liposomal formulations of mTHPC in the window-chamber tumor model.

BACKGROUND AND OBJECTIVE: Foslip® and Fospeg® are liposomal formulations of the photosensitizer mTHPC, intended for use in Photodynamic Therapy (PDT) of malignancies. Foslip consists of mTHPC encapsulated in conventional liposomes, Fospeg consists of mTHPC encapsulated in pegylated liposomes. Possible differences in tumor fluorescence and vasculature kinetics between Foslip, Fospeg, and Foscan® were studied using the rat window-chamber model. MATERIAL AND METHODS: In 18 rats a dorsal skin fold window chamber was installed and a mammary carcinoma was transplanted in the subcutaneous tissue. The dosage used for intravenous injection was 0.15 mg/kg mTHPC for each formulation. At seven time-points after injection (5 minutes to 96 hours) fluorescence images were made with a CCD. The achieved mTHPC fluorescence images were corrected for tissue optical properties and autofluorescence by the ratio fluorescence imaging technique of Kascakova et al. Fluorescence intensities of three different regions of interest (ROI) were assessed; tumor tissue, vasculature, and surrounding connective tissue. RESULTS: The three mTHPC formulations showed marked differences in their fluorescence kinetic profile. After injection, vascular mTHPC fluorescence increased for Foslip and Fospeg but decreased for Foscan. Maximum tumor fluorescence is reached at 8 hours for Fospeg and at 24 hours for Foscan and Foslip with overall higher fluorescence for both liposomal formulations. Foscan showed no significant difference in fluorescence intensity between surrounding tissue and tumor tissue (selectivity). However, Fospeg showed a trend toward tumor selectivity at early time points, while Foslip reached a significant difference (P < 0.05) at these time points. CONCLUSIONS: Our results showed marked differences in fluorescence intensities of Fospeg, Foslip, and Foscan, which suggest overall higher bioavailability for the liposomal formulations. Pegylated liposomes seemed most promising for future application; as Fospeg showed highest tumor fluorescence at the earlier time points.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy.

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; approximately 0.2 mm(3) and approximately 10(2) mm(3), respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.

Fluorescence imaging of Foscan and Foslip in the plasma membrane and in whole cells.

A fluorescence microscope equipped with a condenser for total internal reflection (TIR) illumination was combined with a pulsed laser diode and a time-gated image intensifying camera for fluorescence lifetime measurements of single cells. In particular, fluorescence patterns, decay kinetics, and lifetime images of the lipophilic photosensitizers Foscan and Foslip were studied in whole cells as well as in close vicinity to their plasma membranes. Fluorescence lifetimes of both photosensitizers in cultivated HeLa cells decreased from about 8 ns at an incubation time of 3 h to about 5 ns at an incubation time of 24 h. This seems to result from an increase in aggregation (or self-quenching) of the photosensitizers during incubation. Selective measurements within or in close proximity to the plasma membrane indicate that Foscan and Foslip are taken up by the cells in a similar way, but may be located in different cellular sites after an incubation time of 24 h. A combination of TIR and fluorescence lifetime imaging microscopy (FLIM), described for the first time, appears to be promising for understanding some key mechanisms of photodynamic therapy (PDT).

Ex vivo quantification of mTHPC concentration in tissue: influence of chemical extraction on the optical properties.

A method for the quantification of the concentration of the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) in tissue samples is presented. The technique is an extension of a previously published method based on alkaline hydrolysis of tissue, using Solvable as a tissue solubilizer. mTHPC quantification was achieved by subsequent fluorescence spectroscopy. Since the original extraction method involved multiple steps in which water dilution of the sample was implemented, we studied the spectral characteristics of mTHPC in different Solvable/water mixtures. Using UV-VIS absorption and fluorescence spectroscopy, it was demonstrated that the spectral characteristics of mTHPC vary for different Solvable concentrations. In the range of 20-100% Solvable, the fluorescence intensity of mTHPC did not change, while dramatic changes in the mTHPC fluorescence intensity were observed for lower Solvable concentrations (< 20%) due to increasing hydrophilicity of the environment, combined with pH alterations. We also demonstrated that the absorption and fluorescence spectra of the dissolved tissue were time-dependent. Longer incubation of the samples resulted in a significant increase of the native tissue chromophore fluorescence. This implies that for the correct quantification of photosensitizer concentrations, the fluorescence of native tissue chromophores must be accounted for.

Fluorescence anisotropy imaging reveals localization of meso-tetrahydroxyphenyl chlorin in the nuclear envelope.

We have measured the intrinsic fluorescence anisotropies of six photosensitizers in homogeneous solution, and we have imaged the anisotropies of these sensitizers in tumor cell monolayers using polarization-sensitive laser-scanning confocal microscopy. The intrinsic anisotropies are unremarkable and are within the approximate range of 0.2-0.27. In cells, however, very interesting behavior is exhibited by meso-tetrahydroxyphenyl chlorin (mTHPC). Polarization-sensitive images of mTHPC's fluorescence show a pronounced banding of alternating high and low anisotropy consistent with an ordering of the sensitizer in the nuclear envelope, indicating that this structure is a target of photodynamic damage with this sensitizer. None of the other sensitizers exhibits localization to the nuclear envelope. The frequency distributions of the intracellular anisotropies of the sensitizers exhibit variable peaks and widths. An unusual case is that of Photofrin, with a peak in its anisotropy frequency distribution of -0.12. The change from a positive intrinsic anisotropy in homogeneous solution to a negative value in cells suggests an environmentally induced change in the relative orientations of the absorption and emission dipole moments.

HPLC-electrospray mass spectrometry for the analysis of temoporfin-poly(ethylene glycol) conjugates.

The photodynamic therapeutic agent temoporfin, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) conjugated with poly(ethylene glycol) 2000 (PEG), has been analysed by high performance liquid chromatography (HPLC), linked to electrospray ionisation mass spectrometry (ESI-MS). Sufficient separation of m-THPC-PEG 2000 oligomers was achieved, enabling determination of molecular mass. The use of ESI-MS alone could not achieve this, because of too great a complexity in the mass spectrum, resulting from the presence of four PEG 2000 side chains with a wide molecular mass distribution. The technique is applicable to similar PEG conjugated compounds.

Sensitive isocratic liquid chromatographic assay for the determination of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection.

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C18 column (70 x 4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5-2000 and 10-1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.

Laser-induced fluorescence studies of meso-tetra(hydroxyphenyl)chlorin in malignant and normal tissues in rats.

meso-Tetra(hydroxyphenyl)chlorin (mTHPC) is an attractive second-generation dihydroporphyrin photosensitiser for use in photodynamic therapy. In this study, 1.3 mg kg-1 body weight mTHPC was administered intravenously, and laser-induced fluorescence was used to characterise and compare its localisation and retention in different rat tissues, including an induced experimental adenocarcinoma, 24 h and 48 h post injection. These studies were performed in an attempt to predict the anatomical locations where mTHPC PDT might be most effective and suggest suitable injection--irradiation intervals in each case. Of particular interest were the intra-abdominal and intrathoracic tissues. The fluorescence was induced at 405 nm and the fluorescence spectrum in the region 450-750 nm was analysed. All collected spectra were dominated by the fluorescence signature of mTHPC with its peak at 652 nm, and all values in this study are in terms of background-free drug-specific fluorescence intensity at that wavelength. The photosensitiser accumulated in high concentrations in the tumour and the reticuloendothelial system. Muscular organs, such as the heart and the abdominal wall, were characterised by a low drug fluorescence signature.

Determination of 5,10,15,20-tetra-(m-hydroxyphenyl)chlorin in tissues by high performance liquid chromatography.

A procedure for the extraction and high performance liquid chromatographic (HPLC) determination of the photodynamic therapeutic agent 5,10,15,20-tetra(m-hydroxyphenyl)chlorin in human, rat and mouse tissues following intravenous administration of the drug is described. The tissue (tumour, skin, muscle and liver) was homogenized and extracted into a mixture of methanol:dimethyl sulphoxide:water (32:8:1 by vol.) containing, 5,10,15,20-tetra(p-hydroxyphenyl)chlorin as the internal standard. The precipitated proteins were removed by centrifugation and the supernatant was separated by reversed phase HPLC on a Hypersil-ODS column with 77% (v/v) acetonitrile in 0.1% trifluoroacetic acid as the mobile phase. The solute was detected with high sensitivity and specificity by a UV-VIS detector set at 423 nm.

Model for the porphyrin-DNA binding site: ENDOR investigations of Cu-porphyrins binding to DNA.

Proton ENDOR has been observed from frozen solutions (ca. 38K degrees) of copper meso-(4-N-tetra-methylpyridyl)porphyrin (CuTMpyP(4)) complexed with Salmon sperm DNA in water and D2O. Lines from exchangeable protons of the DNA bases have been observed in these ENDOR spectra. Analyses of these ENDOR data show that the separations of these DNA protons from the copper atom are between 3.76 and 3.84 A with angles of 19.5 to 22.5 degrees between the Cu-H vectors and the gz axis. A distant ENDOR response has also been observed from phosphorous nuclei in the DNA backbone. We estimate that the phosphorous atoms producing this ENDOR signal are 7.5-10 A from the copper center of the porphyrin. These ENDOR data combined with results from an earlier NMR investigation have been used to construct a computer simulated model of the binding site in which the porphyrin is partially intercalated and extends into the major groove of DNA. The two GC base pairs at this site are slightly inequivalent. For each, the G imino proton and one of the C amino protons are at appropriate positions to account for the ENDOR signals arising from exchangeable protons. It is unlikely that this inequivalence would persist at room temperature where dynamic processes would give an apparently symmetric interaction. Although the model accounts for all reported experimental data involving tetracationic porphyrin species which have been suggested to be intercalators, it is not a unique solution.

High-performance liquid chromatographic analysis of porphyrins in clinical materials.

Methods for the isolation of porphyrins as their methyl esters from porphyric urine and faeces as well as other biological materials are described. Quantitative analyses can be carried out by high-performance liquid chromatography (HPLC), using appropriate internal standards; hence excretion patterns in the various types of porphyria can be obtained which may facilitate clinical diagnosis more effectively than the earlier qualitative thin-layer chromatographic methods. Use of the newer microparticulate column packing materials has improved the efficiency of the HPLC analyses, and enables the more convenient isochratic elution techniques to be used (rather than gradient elution). Separations of some porphyrin isomers on these columns are also described.