Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 146 (2015) 249–254
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Physicochemical properties of potential porphyrin photosensitizers for photodynamic therapy Marta Kempa a,b,⇑, Patrycja Kozub a,b, Joseph Kimball c, Marcin Rojkiewicz d, Piotr Kus´ d, Zugmunt Gryczyn´ski c, Alicja Ratuszna a,b a
Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland A. Chełkowski Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland Department of Physics & Astronomy, Texas Christian University, Fort Worth, TX 76129, USA d Institute of Chemistry, University of Silesia, Szkolna 9, 40-006 Katowice, Poland b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Measurements of absorption and
emission spectra of photosensitizers. Quantum yields of fluorescence and
singlet oxygen generation by dyes. Determination of the triplet state
lifetime using laser flash photolysis. Determination of fluorescence
lifetimes using TCSPC method. Comparison of results with
commercial compounds.
a r t i c l e
i n f o
Article history: Received 26 October 2014 Received in revised form 24 February 2015 Accepted 8 March 2015 Available online 12 March 2015 Keywords: Photodynamic therapy Porphyrin Singlet oxygen Fluorescence lifetime Triplet state lifetime
a b s t r a c t This research evaluated the suitability of synthetic photosensitizers for their use as potential photosensitizers in photodynamic therapy using steady state and time-resolved spectroscopic techniques. Four tetraphenylporphyrin derivatives were studied in ethanol and dimethyl sulfoxide. The spectroscopic properties namely electronic absorption and emission spectra, ability to generate singlet oxygen, lifetimes of the triplet state, as well as their fluorescence quantum yield were determined. Also time-correlated single photon counting method was used to precisely determine fluorescence lifetimes for all four compounds. Tested compounds exhibit high generation of singlet oxygen, low generation of fluorescence and they are chemical stable during irradiation. The studies show that the tested porphyrins satisfy the conditions of a potential drug in terms of physicochemical properties. Ó 2015 Elsevier B.V. All rights reserved.
Introduction Porphyrins are naturally occurring organic compounds which are involved in a variety of important biological processes. The ⇑ Corresponding author at: Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland. Tel.: +48 32 349 75 52. E-mail addresses: [email protected] (M. Kempa), [email protected] (P. Kozub), [email protected] (J. Kimball), [email protected] (M. Rojkiewicz), [email protected] (P. Kus´), [email protected] (Z. Gryczyn´ski), [email protected] (A. Ratuszna). http://dx.doi.org/10.1016/j.saa.2015.03.076 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
main element of their chemical structure is the porphyrin ring formed from four pyrrole molecules connected by methine bridges @CHA [1]. Depending on the modification of the ring by the lateral substituents, porphyrin derivatives exhibit different spectroscopic properties. All of these compounds have tendency to aggregate, what influences their physicochemical and spectroscopic properties. Porphyrins and their derivatives due to reduced lymphatic drainage, leaky vasculature, high number of LDL receptors, low interstitial pH and large interstitial space tend to favor accumulation in neoplastic lesions comparing to the surrounding normal
250
M. Kempa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 146 (2015) 249–254
tissue [2]. Since they are very effective chromophores of visible light have the ability to produce singlet oxygen and free radicals [3,4]. Due to these unique properties, porphyrins have been successfully used for photodynamic therapy (PDT). PDT is a successful treatment method for cancer and premalignant conditions that leads to the selective destruction of tumor through photodynamic process [5]. This requires a pro-drug to effectively accumulate in diseased tissues. Often a PDT compounds are called photosensitizers since they are activated by light of a particular wavelength corresponding to the absorption band at lowest energy. Subsequently, in the presence of molecular oxygen light induced reactions (photodynamic process) yield cytotoxic products that damage biologically crucial macromolecules and numerous intracellular structures and lead to the destruction of cancer tissue [6,7]. There are two mechanisms causes destruction of tumor cells in the process of oxidation of the biomolecules and leading to the formation of reactive oxygen species (ROS): free radicals (mechanism type I) and singlet oxygen (mechanism type II). Both are induced simultaneously and dominance depends on a variety of factors (including the type of photosensitizer, its concentration, and the concentration of molecular oxygen in the reaction medium) [8–10]. The desired therapeutic effect is related to a number of specific physical, chemical and biological properties of the photosensitizer [8,11]. An ideal photosensitizer should be chemically pure, stable, selectively accumulate in target tissue, have a short time interval between administration and maximum accumulation in the tissue and be rapidly cleared from the body after therapy. The maximum absorption of the photosensitizers should correspond to the optical window between 600 nm and 850 nm [8]. In this optical range tissue penetration is quite high and the energy of triplet state is sufficient enough for singlet oxygen production. It is also important that photosensitizers administered to the patient should have an amphiphilic nature. This ensures that the transportation in the circulatory system takes place without aggregation and with effective penetration through the lipid layer of cell membrane. Furthermore, the photosensitizers should be safe for the patient and their injection must not cause any toxic effects, allergic reactions or other side-effects. An important parameter of the photosensitizer due to its accumulation and subsequent removal from tissue is optimal fluorescence quantum yield. The fluorescence emission may be used to monitor these processes due to its high sensitivity. In order to succeed in the treatment, another key feature is high efficiency of the ROS generation. Most of today’s commonly used dyes are far from being ideal [8–10,12,13]. Such specific requirements for potential photosensitizers used in PDT make difficult to find compounds which would satisfy all of them simultaneously. Furthermore, due to the different morphometric structures of various neoplasms (presence of connective tissue, tumor vasculature, etc.), a single dye cannot be effective for treating different types of cancer. Therefore, it is important to search for new photosensitizers which would have the desired properties for photodynamic therapy. Depending on the environment in which the test compound is dissolved its photophysical properties may change. In most cases shift of the absorption bands, change in their shape, intensity and tendency to aggregate are observed. Determination of the effect of the environment on test compounds is essential in photodynamic therapy process. This allows to predict how the photosensitizer will behave in contact with other compounds in the cell [14,15]. The aim of the study was to determine the physicochemical characteristics of the four compounds from the porphyrin group as potential photosensitizers used for PDT. These compounds are derivatives of tetraphenylporphyrin (TPP): 5,10,15,20-tetrakis(3hydroxyphenyl)-porphyrin (Porphyrin 1); 5,10,15,20-tetrakis
Fig. 1. Structures of tested porphyrins.
(4-hydroxyphenyl)-porphyrin (Porphyrin 2); 5,10,15,20-tetrakis (3-methoxy-4-hydroxyphenyl)-porphyrin (Porphyrin 3) and 5,10,15, 20-tetrakis(3,5dimethoxy-4-hydroxyphenyl)-porphyrin (Porphyrin 4). Their structures are shown in Fig. 1. We evaluated the effect of different substituents and various polar environments (ethanol and DMSO) on spectroscopic parameters that we expect are crucial role for their PDT properties. In our previous publication we have estimated certain parameters in toluene, chloroform and methanol [16]. Additionally measured parameters and collected information allowed us to draw conclusions regarding the usefulness and effectiveness of tested compounds in photodynamic therapy. The obtained results will be compared against a commercial photosensitizer – TPP. This compound is not applied in photodynamic therapy, but it is widely used as a standard in conducting experiments regarding to determine the physicochemical properties of photosensitizers. Experimental Materials Tested compounds are tetraphenylporphyrin derivatives and were synthesized at the Institute of Chemistry University of Silesia. Detailed description of the synthesis is presented in Rojkiewicz et.al. [16]. 5,10,15,20-tetraphenyl-21H,23H-porphyrin (TPP), phenalenone (Phe) and Ludox were purchased from Sigma Aldrich Company. Ethanol, dimethyl sulfoxide (DMSO) and toluene were obtained from POCH S.A. Methods Absorption, emission and excitation spectra Absorption spectra were recorded using Hitachi UV–VIS spectrophotometer U-1900. Spectra were collected from 300 nm to 800 nm in 2 nm steps. Emission and excitation spectra were obtained using Hitachi F-7000 spectrofluorometer from 550 nm to 800 nm and 300 nm to 650 nm in 1 nm steps, respectively.
M. Kempa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 146 (2015) 249–254
Spectra were recorded for compounds dissolved in ethanol and DMSO at a concentration 2 lM. Additionally, aggregation was investigated by examining the absorption spectra of the samples in the concentration range from 0.078 lM to 10 lM. All experiments were performed at room temperature using quartz cuvettes. Fluorescence quantum yield To determine the fluorescence quantum yield (/F), a comparative method [17] was used applying TPP dissolved in toluene as a reference [18]. The value of this parameter was calculated using the Eq. (1):
/SF ¼ /RF
aS n2R aR n2S
ð1Þ
where /F – fluorescence quantum yield, n – refractive index of solvent, a – slope of the curve representing the integrated fluorescence intensity as a function of absorbance, S and R refer to the reference and sample, respectively. Measurements were made using Hitachi spectrometers and quartz cuvettes. Experiments were performed at room temperature for air-equilibrated solutions. Fluorescence lifetime Fluorescent emission and lifetime measurements were performed on a FluoTime 300 fluorescence lifetime spectrometer with PicoHarp 300 TCSPC Module (Piqoquant, GmbH). Pulsed laser excitation for lifetime measurements provided by a LDH-D-C-485 (Piqoquant, GmbH) with spectral FWHM 485 ± 5 nm and pulse width Int [ns]
650 < s > Amp [ns]
720 < s > Int [ns]
720 < s > Amp [ns]
P1 P2 P3 P4 TPP
10.04 9.95 9.24 9.41 10.48
0.67 0.40 0.60 0.27 0.82
0.91 0.35 0.56 0.24 0.75
7.81 8.37 7.27 7.25 7.98
0.33 0.60 0.40 0.73 0.18
0.09 0.65 0.44 0.76 0.25
0.97 0.95 0.97 0.96 1
0.91 0.98 1.03 0.99 1
9.31 9.01 8.43 7.83 10.03
9.18 8.95 8.32 7.72 9.93
9.84 8.92 8.37 7.77 9.87
9.79 8.86 8.25 7.67 9.73
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M. Kempa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 146 (2015) 249–254
photodegradation tests showed that these porphyrins are characterized by high stability. During exposure of the compounds to red light, no changes were observed in the absorption spectrum (data not presented). Thereby, the photosensitizer will not be degraded during irradiation of the tumor, and thus will not reduce the effectiveness of PDT. Conclusion
[3] [4] [5] [6] [7] [8] [9] [10] [11]
We have presented spectroscopic and physicochemical properties of four derivative porphyrins. Test compounds differ in their types of substituents (hydroxyl and methoxyl groups) and their positions on the phenyl group. Characterization of the porphyrin derivatives’ physicochemical properties is important due to the design of new synthetic photosensitizers. This study shows that the use of such substitutions do not significantly affect their optical spectra. Their high photostability also indicates these compounds as strong candidates for PDT due to their stability under prolonged irradiation required for PDT. The determined lifetimes of the excited triplet state of the photosensitizers are typical for porphyrin group. Residence of the molecule in the metastable triplet state allows an efficient energy transfer to molecular oxygen. In accordance with the requirements of an ideal photosensitizer, the lifetime of triplet states should be of the order of microseconds [44], what was obtained in our experiment. As expected, the tested compounds exhibited high singlet oxygen yield generation required for PDT. The determined fluorescence lifetimes of these photosensitizers are comparable relative to each other. They exhibit low (15%) fluorescence quantum yield. However, these values are sufficient to use photosensitizers as a tool for diagnosis or monitoring of photosensitizer accumulation in the body. In comparison to the commercial photosensitizer TPP, these phorphyrins were characterized by better physicochemical parameters related to their use as anticancer compounds in photodynamic therapy. At this stage of the study, these photosensitizers have been determined to exhibit the required conditions to become used in photodynamic therapy. For the future verification, further in vitro and in vivo tests are required. Acknowledgments The work of two authors (Patrycja Kozub and Marta Kempa) was partially supported by the scholarships within the framework of the TWING project co-financed by the European Social Fund. References
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Physicochemical properties of potential porphyrin photosensitizers for photodynamic therapy Marta Kempa a,b,⇑, Patrycja Kozub a,b, Joseph Kimball c, Marcin Rojkiewicz d, Piotr Kus´ d, Zugmunt Gryczyn´ski c, Alicja Ratuszna a,b a
Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland A. Chełkowski Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Poland Department of Physics & Astronomy, Texas Christian University, Fort Worth, TX 76129, USA d Institute of Chemistry, University of Silesia, Szkolna 9, 40-006 Katowice, Poland b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Measurements of absorption and
emission spectra of photosensitizers. Quantum yields of fluorescence and
singlet oxygen generation by dyes. Determination of the triplet state
lifetime using laser flash photolysis. Determination of fluorescence
lifetimes using TCSPC method. Comparison of results with
commercial compounds.
a r t i c l e
i n f o
Article history: Received 26 October 2014 Received in revised form 24 February 2015 Accepted 8 March 2015 Available online 12 March 2015 Keywords: Photodynamic therapy Porphyrin Singlet oxygen Fluorescence lifetime Triplet state lifetime
a b s t r a c t This research evaluated the suitability of synthetic photosensitizers for their use as potential photosensitizers in photodynamic therapy using steady state and time-resolved spectroscopic techniques. Four tetraphenylporphyrin derivatives were studied in ethanol and dimethyl sulfoxide. The spectroscopic properties namely electronic absorption and emission spectra, ability to generate singlet oxygen, lifetimes of the triplet state, as well as their fluorescence quantum yield were determined. Also time-correlated single photon counting method was used to precisely determine fluorescence lifetimes for all four compounds. Tested compounds exhibit high generation of singlet oxygen, low generation of fluorescence and they are chemical stable during irradiation. The studies show that the tested porphyrins satisfy the conditions of a potential drug in terms of physicochemical properties. Ó 2015 Elsevier B.V. All rights reserved.
Introduction Porphyrins are naturally occurring organic compounds which are involved in a variety of important biological processes. The ⇑ Corresponding author at: Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland. Tel.: +48 32 349 75 52. E-mail addresses: [email protected] (M. Kempa), [email protected] (P. Kozub), [email protected] (J. Kimball), [email protected] (M. Rojkiewicz), [email protected] (P. Kus´), [email protected] (Z. Gryczyn´ski), [email protected] (A. Ratuszna). http://dx.doi.org/10.1016/j.saa.2015.03.076 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
main element of their chemical structure is the porphyrin ring formed from four pyrrole molecules connected by methine bridges @CHA [1]. Depending on the modification of the ring by the lateral substituents, porphyrin derivatives exhibit different spectroscopic properties. All of these compounds have tendency to aggregate, what influences their physicochemical and spectroscopic properties. Porphyrins and their derivatives due to reduced lymphatic drainage, leaky vasculature, high number of LDL receptors, low interstitial pH and large interstitial space tend to favor accumulation in neoplastic lesions comparing to the surrounding normal
250
M. Kempa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 146 (2015) 249–254
tissue [2]. Since they are very effective chromophores of visible light have the ability to produce singlet oxygen and free radicals [3,4]. Due to these unique properties, porphyrins have been successfully used for photodynamic therapy (PDT). PDT is a successful treatment method for cancer and premalignant conditions that leads to the selective destruction of tumor through photodynamic process [5]. This requires a pro-drug to effectively accumulate in diseased tissues. Often a PDT compounds are called photosensitizers since they are activated by light of a particular wavelength corresponding to the absorption band at lowest energy. Subsequently, in the presence of molecular oxygen light induced reactions (photodynamic process) yield cytotoxic products that damage biologically crucial macromolecules and numerous intracellular structures and lead to the destruction of cancer tissue [6,7]. There are two mechanisms causes destruction of tumor cells in the process of oxidation of the biomolecules and leading to the formation of reactive oxygen species (ROS): free radicals (mechanism type I) and singlet oxygen (mechanism type II). Both are induced simultaneously and dominance depends on a variety of factors (including the type of photosensitizer, its concentration, and the concentration of molecular oxygen in the reaction medium) [8–10]. The desired therapeutic effect is related to a number of specific physical, chemical and biological properties of the photosensitizer [8,11]. An ideal photosensitizer should be chemically pure, stable, selectively accumulate in target tissue, have a short time interval between administration and maximum accumulation in the tissue and be rapidly cleared from the body after therapy. The maximum absorption of the photosensitizers should correspond to the optical window between 600 nm and 850 nm [8]. In this optical range tissue penetration is quite high and the energy of triplet state is sufficient enough for singlet oxygen production. It is also important that photosensitizers administered to the patient should have an amphiphilic nature. This ensures that the transportation in the circulatory system takes place without aggregation and with effective penetration through the lipid layer of cell membrane. Furthermore, the photosensitizers should be safe for the patient and their injection must not cause any toxic effects, allergic reactions or other side-effects. An important parameter of the photosensitizer due to its accumulation and subsequent removal from tissue is optimal fluorescence quantum yield. The fluorescence emission may be used to monitor these processes due to its high sensitivity. In order to succeed in the treatment, another key feature is high efficiency of the ROS generation. Most of today’s commonly used dyes are far from being ideal [8–10,12,13]. Such specific requirements for potential photosensitizers used in PDT make difficult to find compounds which would satisfy all of them simultaneously. Furthermore, due to the different morphometric structures of various neoplasms (presence of connective tissue, tumor vasculature, etc.), a single dye cannot be effective for treating different types of cancer. Therefore, it is important to search for new photosensitizers which would have the desired properties for photodynamic therapy. Depending on the environment in which the test compound is dissolved its photophysical properties may change. In most cases shift of the absorption bands, change in their shape, intensity and tendency to aggregate are observed. Determination of the effect of the environment on test compounds is essential in photodynamic therapy process. This allows to predict how the photosensitizer will behave in contact with other compounds in the cell [14,15]. The aim of the study was to determine the physicochemical characteristics of the four compounds from the porphyrin group as potential photosensitizers used for PDT. These compounds are derivatives of tetraphenylporphyrin (TPP): 5,10,15,20-tetrakis(3hydroxyphenyl)-porphyrin (Porphyrin 1); 5,10,15,20-tetrakis
Fig. 1. Structures of tested porphyrins.
(4-hydroxyphenyl)-porphyrin (Porphyrin 2); 5,10,15,20-tetrakis (3-methoxy-4-hydroxyphenyl)-porphyrin (Porphyrin 3) and 5,10,15, 20-tetrakis(3,5dimethoxy-4-hydroxyphenyl)-porphyrin (Porphyrin 4). Their structures are shown in Fig. 1. We evaluated the effect of different substituents and various polar environments (ethanol and DMSO) on spectroscopic parameters that we expect are crucial role for their PDT properties. In our previous publication we have estimated certain parameters in toluene, chloroform and methanol [16]. Additionally measured parameters and collected information allowed us to draw conclusions regarding the usefulness and effectiveness of tested compounds in photodynamic therapy. The obtained results will be compared against a commercial photosensitizer – TPP. This compound is not applied in photodynamic therapy, but it is widely used as a standard in conducting experiments regarding to determine the physicochemical properties of photosensitizers. Experimental Materials Tested compounds are tetraphenylporphyrin derivatives and were synthesized at the Institute of Chemistry University of Silesia. Detailed description of the synthesis is presented in Rojkiewicz et.al. [16]. 5,10,15,20-tetraphenyl-21H,23H-porphyrin (TPP), phenalenone (Phe) and Ludox were purchased from Sigma Aldrich Company. Ethanol, dimethyl sulfoxide (DMSO) and toluene were obtained from POCH S.A. Methods Absorption, emission and excitation spectra Absorption spectra were recorded using Hitachi UV–VIS spectrophotometer U-1900. Spectra were collected from 300 nm to 800 nm in 2 nm steps. Emission and excitation spectra were obtained using Hitachi F-7000 spectrofluorometer from 550 nm to 800 nm and 300 nm to 650 nm in 1 nm steps, respectively.
M. Kempa et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 146 (2015) 249–254
Spectra were recorded for compounds dissolved in ethanol and DMSO at a concentration 2 lM. Additionally, aggregation was investigated by examining the absorption spectra of the samples in the concentration range from 0.078 lM to 10 lM. All experiments were performed at room temperature using quartz cuvettes. Fluorescence quantum yield To determine the fluorescence quantum yield (/F), a comparative method [17] was used applying TPP dissolved in toluene as a reference [18]. The value of this parameter was calculated using the Eq. (1):
/SF ¼ /RF
aS n2R aR n2S
ð1Þ
where /F – fluorescence quantum yield, n – refractive index of solvent, a – slope of the curve representing the integrated fluorescence intensity as a function of absorbance, S and R refer to the reference and sample, respectively. Measurements were made using Hitachi spectrometers and quartz cuvettes. Experiments were performed at room temperature for air-equilibrated solutions. Fluorescence lifetime Fluorescent emission and lifetime measurements were performed on a FluoTime 300 fluorescence lifetime spectrometer with PicoHarp 300 TCSPC Module (Piqoquant, GmbH). Pulsed laser excitation for lifetime measurements provided by a LDH-D-C-485 (Piqoquant, GmbH) with spectral FWHM 485 ± 5 nm and pulse width Int [ns]
650 < s > Amp [ns]
720 < s > Int [ns]
720 < s > Amp [ns]
P1 P2 P3 P4 TPP
10.04 9.95 9.24 9.41 10.48
0.67 0.40 0.60 0.27 0.82
0.91 0.35 0.56 0.24 0.75
7.81 8.37 7.27 7.25 7.98
0.33 0.60 0.40 0.73 0.18
0.09 0.65 0.44 0.76 0.25
0.97 0.95 0.97 0.96 1
0.91 0.98 1.03 0.99 1
9.31 9.01 8.43 7.83 10.03
9.18 8.95 8.32 7.72 9.93
9.84 8.92 8.37 7.77 9.87
9.79 8.86 8.25 7.67 9.73
254
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photodegradation tests showed that these porphyrins are characterized by high stability. During exposure of the compounds to red light, no changes were observed in the absorption spectrum (data not presented). Thereby, the photosensitizer will not be degraded during irradiation of the tumor, and thus will not reduce the effectiveness of PDT. Conclusion
[3] [4] [5] [6] [7] [8] [9] [10] [11]
We have presented spectroscopic and physicochemical properties of four derivative porphyrins. Test compounds differ in their types of substituents (hydroxyl and methoxyl groups) and their positions on the phenyl group. Characterization of the porphyrin derivatives’ physicochemical properties is important due to the design of new synthetic photosensitizers. This study shows that the use of such substitutions do not significantly affect their optical spectra. Their high photostability also indicates these compounds as strong candidates for PDT due to their stability under prolonged irradiation required for PDT. The determined lifetimes of the excited triplet state of the photosensitizers are typical for porphyrin group. Residence of the molecule in the metastable triplet state allows an efficient energy transfer to molecular oxygen. In accordance with the requirements of an ideal photosensitizer, the lifetime of triplet states should be of the order of microseconds [44], what was obtained in our experiment. As expected, the tested compounds exhibited high singlet oxygen yield generation required for PDT. The determined fluorescence lifetimes of these photosensitizers are comparable relative to each other. They exhibit low (15%) fluorescence quantum yield. However, these values are sufficient to use photosensitizers as a tool for diagnosis or monitoring of photosensitizer accumulation in the body. In comparison to the commercial photosensitizer TPP, these phorphyrins were characterized by better physicochemical parameters related to their use as anticancer compounds in photodynamic therapy. At this stage of the study, these photosensitizers have been determined to exhibit the required conditions to become used in photodynamic therapy. For the future verification, further in vitro and in vivo tests are required. Acknowledgments The work of two authors (Patrycja Kozub and Marta Kempa) was partially supported by the scholarships within the framework of the TWING project co-financed by the European Social Fund. References
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