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Dose estimation of pyropheophorbide Methyl Ester and pyropheophorbide a in vitro under oxygenated condition.


Many photosensitizers used in biomedical studies are degraded upon light illumination. This process, usually called photobleaching or phoptodegradation that results in a decrease in the absorption and fluorescence intensities. Investigations have been performed on photobleaching compounds like chlorins and phthalocyanines in vitro and in vivo (Spikes and Bommer, 1993; Streckyte and Rotomskis, 1993). In these studies, it was shown that different sensitizers photodegrade at different rates in solutions, cells, and tissues. Upon illumination hematoporphyrin or its derivatives visible-absorbing photoproducts were observed by some researchers (Mang et al., 1987; Moan and Kessel, 1988). The photosensitizers, when irradiated, are probably attacked by the singlet oxygen [sup.1][O.sub.2] ([sup.1][[summation].sup.-.sub.g]]) they produce, although free radicals may also play a role (Feix and Kalyanaraman, 1991; Baker and Kanofsky, 1992).

When the sensitizer is too fast bleached during illumination, the cancerous cells may not be destroyed completely (Moan, 1986). On other side, the photobleaching of the sensitizer can result in reducing its concentration in the target tissue, especially in the skin and decrease the level of irreversible damage to normal tissue (Boyle and Potter, 1987). Additional information on the photobleaching behaviour of the photosensitizers is needed to calculate the optimum photodynamic therapy (PDT) dose. Thus, a scientific basis of calculation the PDT dose can be provided by studying the photobleaching of sensitizers (Potter et al., 1987).

Pyropheophorbide methyl ester (PPME), as with other sensitizers, induced a biological damage depends on the singlet oxygen generation and other radicals of the reactive species of the molecular oxygen, (MacDonald et al., 1999; Al-Omari et al., 2008; Kanony et al., 2003) and thus depends on the availability of molecular oxygen in its triplet ground state and the illumination of the treatment light. Since the photosensitizer concentration, oxygen, and the light irradiation are difficult to measure in vivo during treatment, incorporating these measures into an explicit dose is not easy. On the other hand, PDT dose estimations is essentially based on either a direct or indirect measure of molecular singlet oxygen (Fontaine-Aupart et al., 1995).

In this study, we investigated the photobleaching of pyropheophorbide methyl ester (PPME) and pyropheophorbide a (PPa) in a homogeneous solution of dimethylformamide (DMF) during PDT and estimated whether fluorescence photobleaching of the sensitizer or the formation of its photoproduct is appropriate for evaluating PDT dose.

Materials and methods


From Sigma-Aldrich the pyropheophorbide methyl ester was purchased. The compounds were used and kept in the dark at low temperature. Pyropheophorbide (PPa) was taken as a reference compound (Al-Omari and Ali, 2009). The structural formula of PPa and PPME are shown in Fig. 1.


Absorption spectroscopy

Using spectrophotometer Shimadzu UV-1700 the electronic ground-state absorption spectra were recorded at the room temperature. The increment of the measurements was 0.1 nm.

Steady-state fluorescence spectroscopy

The steady-state fluorescence spectra were registered on JASCO-FP 6500 spectroscopy using fluorescence quartz cell (1 cm x 1 cm path length) at room temperature. The parameters were constant for each sample enabling a maximum intensity of less than151,000 counts. The excitation wavelength was 412 nm.

Photobleaching measurements

Upon illumination 2.5 ml samples placed in a 1 x 1 cm quartz cell using He-Ne laser (light irradiance: 23 mWcrrT2, emission wavelength: 632 nm), phototransformation was produced. The heat effect was reduced using a water path. The steady-state fluorescence was perpendicularly detected to the direction of excitation in an L-shaped setup. For all samples, the concentrations were about 2 [micro]M at the excitation wavelength. The steady-state fluorescence spectra were frequently recorded for 130 minutes to observe the photodestruction. The absorption spectra were registered before and after illumination.

A photosensitizer with a concentration [H] undergoes several deactivation processes of the excited state upon a continuous stationary irradiation. The process can be expresses by (Fontaine- Aupart et al., 1995)

d[H]/dt = -I(t)[K.sub.FL][H] (1)

where, I(t) refers to the incident light fluence rate absorbed by the photosensitizer, and [K.sub.Fl] is the fluorescence bleaching constant of the investigated sensitizer. Depending on the definition of the optical density (OD = [epsilon][H]l, where l is the cell length, and [epsilon] is the extinction coefficient) the concentration [H] in the above equation can be replaced by the optical density OD.

The constant of the fluorescence bleaching ([K.sub.Fl]) for the molecules under investigation can be calculated by the following relationship (Al-Omari and Ali, 2009)

[K.sub.Fl] = [DELTA]F(t)/[F.sub.0] 1/O[D.sub.exc] 1/D(t) (2)

where, F(t) is the area of the fluorescence spectrum at an illumination time of t, [F.sub.0] is the integrated area under the fluorescence spectrum at the illumination time of zero, O[D.sub.exc] refers to the absorbance at the excitation wavelength, and the incident illumination dose (D) which is the multiplication of the incident light fluence rate I(t) and irradiation time (t).


Solutions of PPME in DMF followed the Beer-Lambert law up to 46 [micro]M, indicating that under such conditions PPME is monomeric. The electronic absorption spectra of PPa and PPME in DMF are shown in Fig. 2. As seen, the shape of the absorption spectrum of PPME resembles that of PPa with two strong absorption vibronic bands centered at 412.5 nm (belongs to B band) with molar extinction coefficient of [epsilon]412.5nm = 1.15 x [10.sup.5] [M.sup.-1] [cm.sup.-1] and at 666.8 nm (belongs to Q bands) with molar extinction coefficient of [epsilon]666.8nm = 4.96 x [10.sup.4] [M.sup.-1] [cm.sup.-1]. The Q bands consist of other three weaker maxima positioned at 508, 537 and 609 nm, respectively.


For the investigated molecules the fluorescence spectra are shown in Fig. 3. They have similar shapes. Despite this, for PPME the maximum peak ([[lambda].sub.max] = 675 nm) is hypsochromic shift with regard to that of PPa ([[lambda].sub.max] = 676 nm).

A reduction in the absorption and fluorescence intensities occur with no new band appearance or bands shift over whole spectral regions upon illumination PPa and PPME in DMF solvent. The reduction is monotonic with increasing the illumination time. The intensity, spectral position, and shape of the vibronic bands were the same after 130 minutes of illumination. The photobleaching efficiency of PPa is stronger than that of PPME.



From the point of view of its potential application in the PDT of cancer pyropheophorbide a methyl ester (PPME) is a therapeutic second generation photosensitizer (Al-Omari and Ali, 2009). It belongs to the family of porphyrins. They are known to be photobleached when exposed to a light and used in photosensitized tumor therapy (Moan, 1986). In general, as compared with other porphyrins the pyropheophorbide derivatives display absorption vibronic bands at longer wavelengths exhibiting higher molar extinction coefficients (Lin, 1991), (see, Fig. 2). The great importance of such excellent spectroscopic properties is that they give a higher efficiency of light absorption and subsequent photosensation.

The fluorescence quantum yield of the investigated molecules are reduced during illumination. The absorbance or fluorescence decreases linearly with increasing the illumination time. After 130 minutes the fluorescence quantum yields were 0.30 and 0.12 less than their initial values of PPME and PPa, respectively. In addition, no recovery in the dark has been detected when recording the absorption as well as fluorescence spectra after two days. The photobleaching efficiency of PPa ([K.sup.PPa.sub.Fl] =14 x [10.sup.-3] [cm.sup.2]/J) is higher than that of PPME ([K.sup.PPPME.sub.Fl] = 5 x [10.sup.-3] [cm.sup.2]/J) as presented in Table 1. The singlet oxygen quantum yield ([[PHI].sub.[DELTA]]) for both compounds have the similar trend. That is for PPa it is [[PHI].sup.PPa.sub.[DELTA]] = 0.52% and it is only [[PHI].sup.PPME.sub.[DELTA]] = 0.19 for PPME (Al-Omari et al., 2008) (Table 1). This might be interpreted as following; generally the photostability of the molecular systems depends strongly on their intersystem crossing quantum yield and hence on their singlet oxygen quantum yields (Fiedor et al., 2002). Therefore, the photosensitizer PPa with the highest singlet oxygen quantum yield would have the higher photobleaching constant. On the contrary, the PPME molecule with low singlet oxygen quantum yield would have the least photobleaching constant. Therefore, the singlet oxygen is probably the main kind of the reactive oxygen species which is responsible for photobleaching of PPME and PPa. Moreover, when comparing the ratio of their photobleaching constants for the tow compounds with the ratio of their singlet oxygen generations; [K.sup.PPa.sub.Fl]= 14 x [10.sup.-3] [cm.sup.2]/J is about 2.8 times greater than [K.sup.PPME.sub.Fl] = 5 x [10.sup.-3][cm.sup.2]/J which is approximately the same ratio of their singlet oxygen quantum yields ([[PHI].sup.PPa.sub.[DELTA]]/[[PHI].sup.PPME.sub.[DELTA]] = 2.7). The slight difference between the two ratios may be attributed to existence of other kinds of reactive oxygen species. Thus, the free radicals such as hydrogen peroxide ([H.sub.2][O.sub.2]), superoxide anion (O2-), and hydroxyl radical (OH-) may be involved in the photobleaching process (Krasnovsky et al., 1990; Georgakoudi et al., 1990). From the view point of improving the efficiency of PDT, the generation of the cytotoxic reactive oxygen species can enhance the therapeutic applications of PPME.

Studying the photosensitizer photobleaching can provide a scientific basis for the calculation of PDT dose (PD) (Moan and Kessel, 1988; MacDonald et al, 1999).

For this purpose, we define the relative critical photobleaching as the dose required for reaching the fluorescence bleaching to [DELTA]F/[F.sub.0] = 0.50 (see, Eq. (2)). Based on this definition, the photodynamic dose of PD = 179 [Jcm.sup.-2] (Table 1) is needed for PPa to bleach a fifty percent of its initial fluorescence. Whereas, for PPME it is required much higher photodynamic dose of PD = 500 Jcmf2 (Table 1) to bleach the same ratio of the fluorescence intensity. This should mean that, under similar conditions of illumination, the photobleaching life time of PPME is about three times longer than that of PPa. The longer photobleaching life time of PPME should be an advantage since the unhealthy tissue is dealt with a less amount of PPME drugs and as a result a less damage of the healthy tissue is obtained.

For a photostable sensitizer with photobleaching constant less than about [10.sup.-2] [J.sup.-1][cm.sup.2], a simple geometrical definition of the photodynamic dose is applied (Potter et al., 1987)

PD = [[H.sub.0]]I (3)

where [[H.sub.0]] is the initial concentration of the photosensitizer ([micro]g[kg.sup.-1]) and I is the incident light dose ([Jcm.sup.-2]). Because the bleaching constant of PPME ([K.sup.PPME.sub.Fl] =5 x [10.sup.-3][cm.sup.2]/J) is less than the above-cited limit, the condition of applying Eq. (3) is applied safely for PPME.

Fig. 4 shows the dependence of the photodynamic dose on the incident light dose for PPME and PPa. As seen, for PPME the curvature trends to be a linear due to the small bleaching constant of 0.005 cm2/J. Therefore, this is another evidence that Eq. (3) is applied conveniently as the bleaching constant reaches 0.01 [cm.sup.2]/J or less.

If the photobleaching is efficient for a sensitizers during the illumination as for PPa (about 50% of the initial fluorescence is bleached at an illumination dose of 179 J/[cm.sup.2]), then, the modified formula of PD is valid (Moan and Kessel, 1988):

PD = [[H.sub.0]]/[K.sub.Fl][1-exp(-[K.sub.Fl]I)] (4)

Eq. (4) is valid for calculating the photodynamic dose of PPa molecule. This is justified when investigating Fig. 4. The curvature of PPa is a clear exponential shape where the photobleaching constant of PPa is 0.014 [cm.sup.2] /J. This is well-correlated with the assumption that states for photobleaching constants higher than 0.01 [cm.sup.2]/J the condition of applying formula (4) is achieved. However, we can show that Eq. (3) is mathematically a special case of Eq. (4) when the magnitude of [K.sub.Fl]I approaches zero. Thus, by applying the limitation on both sides of Eq. (4) we obtain:


= [[H.sub.0]]/[K.sub.Fl][1-)1-[K.sub.Fl] xI)) (6)

= [[H.sub.0]] x I (7)

As seen, Eqs. (3) and (7) are identical. Depending upon this result, we recommend when calculating the photodynamic doses to apply Eq. (4) whether the light dose values small or large.



Pyropheophorbide a (PPa) and pyropheophorbide methyl ester (PPME) were photobleached in dimethylformamide (DMF) upon illumination. The phoptodegradation of PPa is more efficient than that of (PPME) but the photodynamic dose of PPME is greater than that of PPa. Under similar conditions of irradiation, the photobleaching life time of PPME is about three times longer than that of PPa which should be an advantage since the unhealthy tissue is dealt with a less amount of PPME drugs and as a result a less damage of the healthy tissue is obtained.


The authors wish to thank The Deanship of Research and Graduate Studies/Hashemite University for the financial support.


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S. Al-Omari * and A. Ali

Department of Physics, The Hashemite University, Zarqa 13115, Jordan

* Corresponding Author:
Table 1: Photophysical parameters of pyropheophorbide a (PPa) and
pyropheophorbide methyl ester (PPME) in dimethylformamide (DMF) at
room temperature.

Sample [[PHI].sub.[DELTA]] [K.sub.Fl] [x [10.sup.-3 PD [x
 [+ or -] 0.03 [cm.sup.-2] [J.sup.-1]] [Jcm.sup.-2]]

PPME 0.19 5 500
PPa 0.52 14 179
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Author:Al-Omari, S.; Ali, A.
Publication:International Journal of Biotechnology & Biochemistry
Date:Feb 1, 2011
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