Spectroscopic evidence of monomeric aluminium phthalocyanine tetrasulphonate in aqueous solutions

Aqueous solutions of aluminium phthalocyanine tetrasulphonate (AlPcS(4)) were investigated by means of absorption and fluorescence spectroscopy. The absorption spectrum of AlPcS(4) is independent of concentration in a wide range (from 10(-8) to 10(-4) M). The fluorescence spectrum measured with a standard setup is strongly dependent on AlPcS(4) concentration, and the fluorescence maximum is gradually red-shifted with increasing concentration. Calculations that take into account reabsorption of fluorescence (inner-filter effect) fit the experimental observations at low concentrations (up to 10(-6) M). Disagreement between the calculations and spectra recorded at higher concentrations (above 10(-5) M) shows that the reabsorbed light may be reemitted as fluorescence. The influence of inner-filter effects on the spectral shape was demonstrated by the experiments where a fibre-optic front-face fluorescence setup was applied: Under such conditions the shape of the fluorescence spectra for a high concentration (10(-3) M) coincided with that of a low concentration (10(-8) M). In conclusion, the present spectroscopic results show that AlPcS(4) does not form aggregates and is a very stable compound in aqueous solutions.     

Interstitial fluorescence spectroscopy in the human prostate during motexafin lutetium-mediated photodynamic therapy

The in vivo fluorescence emission from human prostates was measured before and after motexafin lutetium (MLu)-mediated photodynamic therapy (PDT). A single side-firing optical fiber was used for both the delivery of 465 nm light-emitting diode excitation light and the collection of emitted fluorescence. It was placed interstitially within the prostate via a closed transparent plastic catheter. Fitting of the collected fluorescence emission spectra using the known fluorescence spectrum of 1 mg/kg MLu in an intralipid phantom yields a quantitative measure of the local MLu concentration. We found that an additional correction factor is needed to account for the reduction of the MLu fluorescence intensity measured in vivo due to strong optical absorption in the prostate. We have adopted an empirical correction formula given by C = (3.1 cm(-1)/micro's) exp (microeff x 0.97 cm), which ranges from approximately 3 to 16, with a mean of 9.3 +/-4.8. Using a computer-controlled step motor to move the probe incrementally along parallel tracks within the prostate we can determine one-dimensional profiles of the MLu concentration. The absolute MLu concentration and the shape of its distribution are confirmed by ex vivo assay and by diffuse absorption measurements, respectively. We find significant heterogeneity in photosensitizer concentration within and among five patients. These variations occur over large enough spatial scales compared with the sampling volume of the fluorescence emission that mapping the distribution in three dimensions is possible.     

Fluorescence imaging during photodynamic therapy of experimental tumors in mice sensitized with disulfonated aluminum phthalocyanine

A fluorescence imaging system was used to monitor the emission of disulfonated aluminum phthalocyanine (AlS2Pc) during the photodynamic therapy (PDT) of murine tumors. Cells of the MS-2 fibrosarcoma were injected in mice in two compartments in order to cause the development of tumors in different host tissues. Two drug doses and two uptake times were considered. Moreover, the fluorescence of the AlS2Pc was excited using two wavelengths on the opposite sides of the absorption peak to detect a possible change in the absorption spectrum of the sensitizer induced by the PDT. In the tumors, the treatment induces a variation of the fluorescence intensity: in some mice a mild photobleaching takes place, in others a fluorescence enhancement occurs. Which effect predominates depends on the experimental conditions, even though a large spread of data was found amongst mice of the same group. In all mice, independently of the drug dose, uptake time or tumor compartment, a marked increase in the fluorescence signal takes place at the borders of the irradiated area. To quantify this effect we evaluated the ratio between the fluorescence intensities in the peritumoral area and in the tumor itself. This ratio increases monotonically during the PDT, showing a different behavior with the two excitation wavelengths. This indicates that the AlS2Pc absorption spectrum shifts toward shorter wavelengths as a result of the irradiation.     

Preparative, analytical and fluorescence spectroscopic studies of sulphonated aluminium phthalocyanine photosensitizers

Fluorescence spectroscopic studies were carried out on aluminium phthalocyanine with defined numbers (mono, di, tri and tetra) of sulphonate groups. Selective sulphonation was achieved using one of two synthetic methods to prepare a mixture of components which were separated using reverse-phase liquid chromatography. Fluorescence lifetimes were measured in methanol and buffer solution using time-correlated single-photon counting with picosecond laser excitation; the lifetime shows little variation with the number of sulphonate groups. Using steady state excitation, fluorescence quantum yields were determined for the tetrasulphonated component (phi F = 0.51) and, for comparison, unsulphonated aluminium phthalocyanine.     

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.     

Association between the photodynamic loss of Bcl-2 and the sensitivity to apoptosis caused by phthalocyanine photodynamic therapy

We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT-induced apoptosis, whether overall cell killing is also sensitized or how up-regulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2delta33-54 and Bcl-2delta37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2delta210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2delta33-54 or Bcl-2delta37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.     

Effects of photodynamic therapy on the absorption properties of disulphonated aluminum phthalocyanine in tumor-bearing mice

Time-resolved reflectance spectroscopy was performed on tumor-bearing mice, administered with disulphonated aluminum phthalocyanine (AlS(2)Pc, 5 mg/kg body weight), before, during and after photodynamic therapy. This allowed us to evaluate the absorption spectrum of AlS(2)Pc in vivo from 610 to 700 nm, and to investigate how the therapeutic irradiation affects it. Two tumor locations (intraderma on the back and intramuscular in the leg), and two uptake times (3 and 12 h) were considered. As already observed previously, the absorption spectrum of AlS(2)Pc in vivo is centered at 680-685 nm. The irradiation causes a blue-shift of the measured line shape, more or less marked depending on the experimental conditions. A reduction in absorption is also often observed upon illumination with therapeutic light doses.     

Tetra-trifluoroethoxyl zinc phthalocyanine: potential photosensitizer for use in the photodynamic therapy of cancer.

Tetra(trifluoroethoxyl) zinc phthalocyanine, which could be dissolved in most organic solvents, was synthesized. The compound displays a good photocytotoxicity on myeloma cells and Chinese hamster ovary cells in vitro in the presence of the emulsifying agent F68.     

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.     

A quantitative comparison of 5-aminolaevulinic acid- and methyl aminolevulinate-induced fluorescence, photobleaching and pain during photodynamic therapy

The characteristics of protoporphyrin IX (PPIX) fluorescence in superficial basal cell carcinoma (sBCC) and carcinoma in situ (Bowen's Disease, BD) following application of 5-aminolaevulinic acid (5-ALA) and its methyl ester (methyl aminolevulinate [MAL]) before, during and after photodynamic therapy (PDT) were investigated in 40 patients. Photosensitizer prodrug penetration can limit PDT efficacy and understanding the characteristics of PPIX fluorescence through fluorescence spectroscopy, may improve knowledge of photosensitizer delivery. Fluorescence intensity was assessed quantitatively, and the rate of photobleaching was determined by fitting an exponential decay. As a secondary end-point, PDT-induced pain was also measured continuously during treatment using a novel hand-held device, known as a pain logger. In vivo PPIX fluorescence was shown to decrease during irradiation, allowing the in vivo photobleaching of PPIX to be monitored. No significant difference was found between ALA- or MAL-induced PPIX fluorescence in lesions of sBCC and BD (P>0.05), indicating no detectable difference in PPIX kinetics for the two prodrugs as assessed by these measures. Pain, as assessed by the logger device, showed high interindividual variability and pain levels tended to be higher initially, decreasing during treatment. No difference was seen in pain experienced during ALA-or MAL-PDT (P>0.05).     

Preparation of a water-soluble fluorinated zinc phthalocyanine and its effect for photodynamic therapy

An amphiphilic fluorinated phthalocyanine, zinc tetracarboxyoctafluorophthalocyanine (ZnC4F8Pc) was synthesized and characterized. Its photodynamic efficiency for HeLa cells was compared with hydrophilic zinc octacarboxyphthalocyanine (ZnC8Pc) and hydrophobic zinc hexadecafluorophthalocyanine (ZnF16Pc). ZnC4F8Pc had a remarkable photodynamic effect among the phthalocyanines used. The effect is apparently caused by the fact that ZnC4F8Pc is mainly accumulated in the hydrophobic lipid membrane and is in the photoactive monomer form in HeLa cells.     

Review of photodynamic therapy for gastrointestinal tumours in the past 6 years in China

Patients (142) with advanced gastrointestinal (GI) cancers were treated by photodynamic therapy (PDT) from September 1982 to December 1988. Haematoporphyrin derivative (HpD) (5 mg kg-1) was intravenously given 48-72 h prior to PDT. The light source was an argon dye laser emitting at 630 nm. The entire tumour was irradiated with a light dose of 100-250 J cm-2. Fifteen patients (10.6%) showed complete response (CR), 53 (37.3%) showed partial response (PR) and 32 (22.5%) showed mild response (MR). The clinical results show that the estimated energy dose (EED) of 200-250 J cm-2 is appropriate. All patients were treated by PDT and adjuvant chemotherapy showing good results with a follow-up of 1-5 years; 13 out of 15 patients in the CR group are alive (86.7%); 12 patients survived more than 2 years (8.8%).     

Long-term effects of photodynamic therapy on fluorescence spectroscopy in the human esophagus.

Clinical interest in laser-induced fluorescence (LIF) spectroscopy and photodynamic therapy (PDT) is growing rapidly and may ultimately lead to close parallel use of these techniques. However, variations in LIF due to photosensitizer retention as well as tissue damage and healing processes may interfere with autofluorescence-based diagnostic methods. We have investigated the compatibility of these two techniques by quantifying PDT-induced changes in LIF in the human esophagus. Fluorescence spectra were collected endoscopically at excitation wavelengths (lambda ex) of 337, 400 and 410 nm in 32 patients. Measurements were performed immediately before and after PDT treatment with porfimer sodium and during follow-up procedures. In the months following PDT regions of reepithelialized squamous showed reduced autofluorescence in comparison with untreated squamous regions (P = 0.0007). Photosensitizer fluorescence was undetectable with lambda ex = 337 nm during follow-up procedures, whereas for lambda ex = 400 and 410 nm porfimer sodium fluorescence was noted for nearly a year after treatment. Therefore, residual photosensitizer fluorescence is likely to affect certain LIF-based diagnostic techniques during a period when patients are at high risk for tumor recurrence. Modification of LIF systems and/or the use of alternative photosensitizers may be required to optimize the detection of lesions in the post-PDT patient. Given the potential of LIF as a method for surveillance following cancer therapy, further investigation of the compatibility of specific LIF approaches with cancer pharmaceuticals may be warranted.     

Control of photobleaching in photodynamic therapy using the photodecarbonylation reaction of ruthenium phthalocyanine complexes via stepwise two-photon excitation

In this study, we have investigated the photochemical properties and photodynamic effects of ruthenium phthalocyanine (RuPc(CO)(Py)) and naphthalocyanine (RuNc(CO)(Py)) complexes. When a nanosecond-pulsed laser is used, the photodecarbonylation of our Ru complexes efficiently proceeds via stepwise two-photon excitation, while the reaction yields are negligibly small when a continuous-wave (CW) laser is employed. The pulsed laser selective photodecarbonylation decreases the Q-band absorbance, which satisfies what the photodynamic therapy (PDT) requires of the photobleaching. For RuPc(CO)(Py), the photochemical reactions including both the photodecarbonylation and just photobleaching occur in HeLa cells in vitro. Toxicity and phototoxicity tests indicate that our RuPc(CO)(Py) and RuNc(CO)(Py) complexes in concentrations of 0.3-1 microM and 1-2 microM, respectively, are applicable as PDT agents. The phototoxicity is consistent with the photochemical properties of these complexes, namely, excited triplet lifetimes (10 and 4.8 micros for the Pc and Nc complexes, respectively) and singlet oxygen yields (0.48 and 0.35 for the Pc and Nc complexes, respectively). On the basis of these results, we propose a novel concept for achieving a greater depth of necrosis in PDT as follows: (1) PDT of upper cellular layers using CW-laser irradiation; (2) efficient photobleaching in upper cellular layers using pulsed dye-laser irradiation, which results in an increase in the therapeutic depth of red light; (3) PDT directed toward deeper tumor tissues using CW laser irradiation. In addition, these Ru complexes are promising as CO release agents for investigative biochemistry.     

Calculation of singlet oxygen dose from photosensitizer fluorescence and photobleaching during mTHPC photodynamic therapy of MLL cells

Predicting the therapeutic outcome of photodynamic therapy (PDT) requires knowledge of the amount of cytoxic species generated. An implicit approach to assessing PDT efficacy has been proposed where changes in photosensitizer (PS) fluorescence during treatment are used to predict treatment outcome. To investigate this, in vitro experiments were performed in which Mat-LyLu cells were incubated in meta-tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 652 nm light. PS concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was monitored during treatment and, at selected fluence levels, cell viability was determined using a colony-formation assay. Singlet oxygen dose was calculated using four different models and was compared with cell survival. For the dose metric based on singlet oxygen-mediated PS photobleaching, a universal relationship between cell survival and singlet oxygen dose was found for all treatment parameters. Analysis of the concentration dependence of bleaching suggests that the lifetime of singlet oxygen within the cell is 0.05-0.25 micros. Generation of about 9 x 10(8) molecules of singlet oxygen per cell reduces the surviving fraction by 1/e.