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As is stated in [1], although the use of photosensitizer photobleaching in implicit dosimetry is attractive in principle, there are a number of issues which must be resolved for it to become routinely useful. The therapeutic outcome of PDT is influenced by sensitizer intratumoral and subcellular distribution. However, the photosensitizers used in PDT demonstrate diffuse localization in many cytoplasmic compartments with the exception of the nucleus [6]. Therefore it would be useful to know the degree of sensitizer coupling in the specific tissue environment, since the absorption properties and other photophysical parameters of sensitizer including photomodification depend on environment.

As is seen in Fig.1

Fig.1. Hp fluorescence spectra in different environment

 the interaction with biological substrate induces changes in the fluorescence emission spectra of Hp. Interaction with HSA causes red shift of Hp emission spectrum. In the emission spectrum of Hp incorporated in ECG the red shift is even more significant and is followed by the broadening of the second emission band. The emission spectrum of Hp in tumour tissue includes both above-mentioned spectral changes. Photobleaching rate of porphyrin molecules localized in protein rich environment would be enhanced due to the interaction of porphyrins with proteins since the proteins accelerate the photobleaching and photoproduct formation of porphyrin-type sensitizers (Fig.2).

Fig.2. Absorption decay curves of Hp registered at the maximum of the third Q band (a); Photoproduct formation curves registered at the absorption maximum of photoproduct (640 nm)

As is seen, the sensitizer-albumin interaction significantly accelerates the photobleaching process, especially at low illumination doses (Fig.2A). Similar tendency is also observed for TPPS4 in PBS of pH 7.2 in the presence of BSA (Fig.3).

Fig.3. Absorption decay curves of TPPS4 in PBS of pH 4.0 and 7.2 and in the presence of BSA

Photobleaching of porphyrin molecules associated with cell membranes would be influenced by membrane components - saturated and unsaturated lipids as well as membrane proteins. Changes in the fluorescence spectra of Hp in ECG suspension are different for Hp localized in ECG membrane and for Hp in aqueous solution (Fig.4).

Fig.4. The changes of Hp fluorescence spectra (C=10-4M) in a suspension of ECG upon illumination. Insert: the fluorescence intensity decay, estimated at 613 and 634 nm.

Free Hp (in solution) which exhibits fluorescence maximum at 613 nm, has lower fluorescence bleaching rate in comparison with the Hp interacted with ECG (which exhibits fluorescence maximum at 634 nm).

From these results it is evident that interaction with proteins or incorporation in ECG membranes accelerates the photomodifiaction process of photosensitizers and the photobleaching rate constant depends on the environment in which the sensitizer is localized. It seems that porphyrin-type sensitizers in cytosol are more photostable than these incorporated in membrane or interacted with proteins. So, the photobleaching rate of photosensitizer and, as a consequence an index of the effective delivered dose in implicit dosimetry in PDT, would depend on the relative distribution of sensitizer between the cytoplasmic compartments. It seems that photobleaching as a parameter for implicit dosimetry could be used only if the corrections would be done regarding the data of the relative amount of sensitizer in certain environment or the photobleaching rate would be estimated as an empirical parameter for the specific sensitizer.

In addition it should be noted that the photobleaching rate will not correlate with the tissue response directly since the response will depend not only on the local oxygen concentration and singlet oxygen generation yield in certain cellular compartment but also on the amount of cellular targets in the close proximity to the site of singlet oxygen generation. Singlet oxygen generated in, for example, a membrane of mitochondria would induce more efficient damage than singlet oxygen generated in cytosol. The local oxygen concentration may be much higher in lipid (membrane) cellular compartments or in protein rich compartments and this would enhance coupled photobleaching rate.

The photobleaching rate of sensitizers is also dependent on pH (Fig.5).

Fig.5. The changes of TPPS4 fluorescence spectra in PBS of pH 7.2 (A) and pH 4.0 (B) upon irradiation

The absorption difference spectra of TPPS4 in PBS at pH 7.2 and pH 4 measured at the illumination with He-Ne laser light (l=632 nm) indicate that depending on pH the different photobleaching rate is observed. At pH 4 only small changes in the Soret band are detected (Fig.5B) contrary to TPPS4 in PBS of pH 7.2 where the photobleaching is noticeably faster (Fig.5A). The photobleaching kinetics normalized to the amount of the absorbed light quanta and the value of initial absorption (see Materials and methods) clearly show that in the acidic environment the sensitizer is more photostable than in neutral (Fig.3). Therefore it should be expected that for such sensitizers as TPPS4 or uropoprphyrin, which are known to localize preferentially in lysosomes [7,8] where pH <5, the fraction of the dye localized in lisosomes would photobleach with different rate from the fraction localized in other cell compartments.

One more problem, which arises considering the photobleaching as a dose metric parameter, is inhomogeneous photobleaching of the fluorescence band of sensitizer during PDT. For example, we have found heterogeneous fluorescence bleaching of ALA-induced PpIX in mice skin under illumination. It might be that PpIX fluorescence band actually consists of the fluorescence bands of few different porphyrins (as proposed by Dietel et al [9]) or the fluorescence of PpIX in vivo slightly differs in different environment similarly as it was detected for Hp in cancerous tissue (Fig.1). The long wavelength wing of the main fluorescence band of ALA-induced PpIX bleaches faster than the short wavelength wing of the fluorescence band, therefore the shift of the fluorescence band to the blue is detected after prolonged illumination at 630 nm (Fig.6).

Fig.6. ALA-induced PpIX fluorescence bleaching in mouse skin

So, different coupling would be expected for different ALA-induced sensitizers or for PpIX localized in different tissue compartments. The degree of coupling makes a significant difference in to how photobleaching measurements should be interpreted and applied as a PDT dose metric.

The above-discussed data concern mostly photobleaching consistent with photodegradation – the conversion of sensitizer into the products that do not absorb visible light significantly. Photobleaching of hematoporphyrin-type sensitizers during illumination is related not only to photodegradation but also is followed by the formation of photoproduct(s) with a new fluorescence band at around 640-670 nm and with increased light absorption in the red spectral region at 640 nm [10-14]. The photoproduct(s) formation may influence sensitization process and therefore also accounts for optimal photodynamic dose [4]. As a rule, the interaction with biological environment enhances photoproduct formation rate. Photoproduct formation kinetics registered at the absorption maximum of photoproduct (640 nm) show that the initial photoproduct formation is faster for Hp in the presence of HSA (Fig.2B).

The red-absorbing photoproduct formed during PDT may have photosensitizing effectiveness and may participate in photodynamic process. On the basis of energy-level diagram indicating photosensitizer photobleaching as implicit dose metric [1] we present a scheme in which the formation of photoproducts induces an additional pathway for singlet oxygen generation (Fig.7).

Fig.7. Energy-level diagram indicating possible porticipation of red absorbing photoproduct in sensitization process

From the dosimetric point of view the photobleaching parameters as a dose metric can only be accepted in the case when all the possible channels inducing biological effect will be involved.

As it was mentioned in [15], the fluorescence of photoproduct formed from ALA-induced PpIX, is only 2% of the pre-PDT PpIX fluorescence - thus it would play a minor role in PDT. In spite of so low fluorescence of photoproducts they might be very efficient in PDT, since, as it is seen in Fig.8,

Fig.8. The changes of the Hp absorption and fluorescence (insert) spectra upon irradiation.

the increase in absorption at 630 nm is almost twofold. However, at the same time the increase in the fluorescence for Hp is only about 5% (Fig.8 insert). So, the fluorescence intensity of photoproduct does not reflect real situation. Absorption changes indicate formation of the significant amount of photoproduct although the fluorescence intensity of photoproduct is very low. From the other side, low fluorescence does not mean low photodynamic efficiency, since the photoproduct may generate singlet oxygen with high yield. Even if the photoproducts are non-active in the generation of cytotoxic species, the increase in the absorption at illumination wavelength (at about 630 nm) could limit the light penetration due to added absorption of the photoproduct in the tissue and cause the shielding effect.