the photosensitizer stabilities of tookad® on aggregation, acidification, and day-light irradiation

Procedia Chemistry 14 ( 2015 ) 474 – 483

1876-6196 © 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer-review under responsibility of the Scientifi c Committee of HK-ICONS 2014 doi: 10.1016/j.proche.2015.03.064

ScienceDirectAvailable online at www.sciencedirect.com

2nd Humboldt Kolleg in conjunction with the International Conference on Natural Sciences, HK-ICONS 2014

The Photosensitizer Stabilities of Tookad® on Aggregation, Acidification, and Day-light Irradiation

Yoga Aji Handokoa,b*, Ferdy Semuel Rondonuwua, Leenawaty Limantarab aGraduate Program of Biology, Satya Wacana Christian University, Diponegoro Str. No. 52–60, Salatiga 50711, Indonesia

bMa Chung Research Center for Photoshyntetic Pigments, Ma Chung University, Villa Puncak Tidar N-1, Malang 65151, Indonesia

Abstract

Tookad® (Pd-Bpheid) as bacteriochlorophylls derivative is third generation of photosensitizer having great promises and efficacy for some cancers treatment. The understanding of the photosensitizer stability in vitro is important and fundamental studies before photodynamic therapy (PDT) pre-clinical treatment, especially their responses to aggregation, acidification, and irradiation. Tookad® stability testing was performed by spectroscopic methods using Varian Cary 50 UV-Vis and Light-Volpi Intralux 1200. The result shows that Tookad® aggregated by water titration and degradation is due to the acidification and day-light irradiation. The day-light irradiation caused Tookad® degradation on Soret and Qy band. The degradation product indicates 3-Acetil-chl and Chlorin. © 2015 Y.A. Handoko, F.S. Rondonuwu, L. Limantara. Published by Elsevier B.V. Peer-review under responsibility of the Scientific Committee of HK-ICONS 2014.

Keywords: Acidification; aggregation; day-light; irradiation; PDT; Tookad®

* Corresponding author. Tel.: +62 815 771 7797; fax: +62 341 550 175 E-mail address: [email protected]

Nomenclature Abs absorbance BChl bacteriochlorophyll Pd-Bpheid Palladium-Bacteriopheoforbide (Tookad®) PDT photodynamic therapy

© 2015 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Peer-review under responsibility of the Scientifi c Committee of HK-ICONS 2014

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Yoga Aji Handoko et al. / Procedia Chemistry 14 ( 2015 ) 474 – 483 475

1. Introduction

In PDT application, there are three main factors considered critical in the PDT method, such as photosensitizer, light, and oxygen1–3 . Observing the effectiveness and performance of its functions, the provision of photosensitizer in PDT applications is usually done through injection2–5 , which in turn the photosensitizer will accumulate in the target tissue for about 3 h to 96 h, according to the type of photosensitizer used6. After the accumulation time is stated sufficient, light fired in the tissues that activated by photosensitizer. In the next stage, the photosensitizer is excited to a triplet by intersystem crossing species that could potentially transfer energy and reacts with oxygen to form singlet oxygen. In this state, the singlet oxygen takes its role to destroy the structure and function of vital cancer cells6.

Basically, BChl is readily degradable into derivatives due to internal factors such as the chemical properties of the pigment, enzyme activity, as well as some external factors, such as light intensity, water, acid, oxygen, temperature, and humidity or a combination of these factors7. The fact in the application, BChl is often found in an aggregated state. The aggregation in vitro can be influenced by the solvent8,9. BChl aggregation caused the effectiveness to descend in PDT application process3,10.

Likewise, BChl molecules can be degraded form of the metal core loss due to the macrocyclic ring in acidic environmental conditions. Metal core release process is referred to as feofitinazation11. In addition, the degradation of BChl molecules can also be caused by irradiation. Phenomenon of irradiation caused the BChl degradation and the formation of degradation products to be known as photodegradation/photobleaching. The changes due to photodegradation may include: breaking a bond, color changes, and the rearrangement of the atoms in a molecule12.

Tookad® is the third generation of BChl derivative sensitizer used in PDT applications13. Tookad® in the chemical structure is the Magnesium as the core BChl which was replaced with Palladium and has removed its phytol group, also known by the name of Palladium bacteriopheoforbide (Pd-BPheid) or WST 09. Currently, PDT applications using Tookad® is still in phase II clinical trials. The clinical trial was conducted on the treatment of prostate cancer12,14. The results of the study reported that Tookad® was able to absorb strongly in the near infrared region and efficient in regenerating oxygen singlet when irradiated and it showed a high level of photostability10. The molecular structure of Tookad® is shown in Figure 1.

Fig. 1. Molecule structure of tookad®

Understanding of the molecular stability in vitro is an important step to study as a fundamental stage in PDT

application. Moreover, in its application, the ability and effectiveness of the photosensitizer showed different responses. Limited information about the molecular character studies in vitro of Tookad® in a solvent, the aggregation effect, acidification, and its interaction with light, make research of the stability of this molecule an interesting study to do. With this understanding, the aim of this research is to examine the effect of aggregation, acidification, and polychromatic light irradiation on the stability of the Tookad® molecule.

476 Yoga Aji Handoko et al. / Procedia Chemistry 14 ( 2015 ) 474 – 483

2. Materials and methods 2.1. Materials and equipment

The materials used were Tookad®, acetone 100 %, methanol 100 %, lactic acid, sulphuric acid, and distilled water. Tookad® was obtained from the Laboratory of the Ludwig-Maximillian University Pigments, Munich, Germany; while acetone, methanol, lactic acid, and sulphuric acid were obtained from Merck®. The tool used was a double beam spectrophotometer Varian Cary 50 UV-Vis embedded with volpi lights Intralux 1200.

2.2. Sample preparation

Tookad® was dissolved in acetone and methanol, then homogenized for 1 min by adding nitrogen gas. The addition of nitrogen gas was intended to avoid contact between Tookad® and oxygen. Furthermore, the Qy absorbance maximum setting was 1 at a wavelength (λ) 300 nm to 1 100 nm. Then, the sample was ready to be used for testing aggregation, acidification, and irradiation of Tookad® molecules.

2.3. Tookad® agregation

Tookad® aggregation was tested by titration of distilled water into the sample which had been prepared with a ratio of acetone/methanol: distilled water: 3.0 : 0.0; 2.0 : 1.0; 1.0 : 2.0; and 0.0 : 3.0.

2.4. Aggregates-acidification of Tookad®

After the Tookad® aggregation conducted, testing aggregates-acidification of the Tookad® molecules done at

each concentration series by adding sulphuric acid and lactic acid separately. The entire sample was measured by using a dual beam spectrophotometer Varian Cary 50 UV-Vis at λ 300 nm to 1 100 nm.

2.5. Tookad® degradation through irradiation

Testing of the molecular degradation of Tookad® by irradiation was performed after Tookad® had been dissolved in acetone and methanol, aggregated with distilled water, and aggregated and added with lactic acid or sulphuric acid concentration in each series. Irradiation was done by using a polychromatic light with intensity 870 lux (lux = 1 lm · m2) at room temperature12. During irradiation, cuvette was closed and stirred.

2.6. Data analysis

Analysis of the data includes profile changes of the spectral pattern, difference spectra, and profile changes of the absorbance value toward time interval due to irradiation. Difference spectra is a profile showing the changing patterns of the spectra after treatment of early spectra. The data were processed using the Origin 6.1. Program and interpreted descriptively.

3. Results and discussion 3.1. Tookad® solubility in acetone and methanol as well as its response to irradiation

Tookad® has molecule characters as other bacteriochorophyll derivatives. Tookad® is a molecule that is sensitive to environmental factors such as solvents, pH changes, light, oxygen, and temperature changes. In solvent, Tookad® has different responses depending on the type of solvent. Tabel 1 shows the Tookad® solubility in acetone and methanol. The different of peak absorption on the third band shows that Tookad® has different interactions in the


solvent. According to Koyama et al.8, Misono et al.9, Limantara et al.15, and Vladkova et al.16, BChl indicates coordination of penta coordinate in acetone and coordination of hexa coordinate in methanol. Coordination types BChl in acetone and methanol is determined by the value of the parameter taft. Parameter taft indicates the strength of the electron donor at a certain level. Solvents which have parameter taft values (β) < 0.5 will form the penta coordination, while solvent which has the parameter taft value (β) > 0.5 will form the hexa coordination8. Thus, acetone which has a parameter taft ~0.48 form the penta coordination while methanol has parameter taft ~0.62 form hexa coordination with BChl molecules. Nishizawa et al.17 and Limantara et al.18 also stated that the coordination between the solvent with BChl molecules will affect the bonding of both atoms in its molecular structure. Acetone forming penta coordination with the core of Magnesium ions causes the molecular structure of BChl to shrink because the bond is only coming from one side of the molecule, while the methanol which forms hexa coordination with the core of Magnesium ions causes BChl structural relaxation because the bond is derived from the two sides of the molecule. The coordination between molecules with solvent represented in absorption peak of the Qx band can affect the stability of the Tookad® molecule.

Table 1. Absorption peak of Tookad® in acetone and methanol

acetone methanol

λ (nm) Abs (A.U) λ (nm) Abs (A.U)

755.04 (Qy) 1.019 755.02 (Qy) 1.011 530.02 (Qx) 0.226 535.00 (Qx) 0.208

385.06 (Soret) 0.480 384.94 (Soret) 0.439

330.05 (Soret) 0.644

Fig. 2. The pattern of spectra changes (A1, B1) and different spectra (A2, B2) of Tookad® against irradiation in acetone (A) and methanol (B)

and its degradation products (P1 and P2) from 0 min to 120 min with time interval of every 20 min.


Figure 2 shows that irradiation resulted in the degradation of Tookad® molecule. Degradation is expressed by a

decrease in absorbance peak at Qx band in acetone and significantly in the Qy band, both in acetone and methanol. Along with the decrease in absorbance at peak of the Qy band, the effects of irradiation also resulted in Tookad® being degraded by light (photobleaching) with the formation of degradation products at wavelength of ~649.99 nm and ~425.08 nm in acetone and ~654.95 nm, ~409.98 nm, and ~345.10 nm to ~349.99 nm in methanol.

A study of the effects of irradiation on some kind of BChl has been done. Limantara et al.10 also proved the photobleaching BChl monomer in methanol and acetone. Furthermore, she reported that photobleaching BChl monomer in methanol is faster than acetone. Thus, this statement supports the results of research into the effects of irradiation on this Tookad® molecule. Through Figure 2 it seems clear that Tookad® degraded faster in methanol than acetone. Profile decrease of the absorption peak in methanol also occurs in BChl degradation studies which were conducted by Limantara et al.19 and chlorophyll by Watanabe et al.19, Jeffrey et al.20,, Fiedor et al.21. They reported that BChl and chlorophyll dissolved in methanol has a value of the oxidation potential lower than the BChl/chlorophyll dissolved in acetone, so the greater degradation in methanol. 3.2. Tookad® aggregation in acetone and methanol as well as its aggregate response to irradiation

The fact in the application is that BChl is often found in the aggregated state. The aggregation can be affected by the solvents, one of which is water. Tookad® are amphiphilic molecules that are hydrophilic on the head, while the tail is phytil group which is hydrophobic so that these molecules do not dissolve in water. According to Katz et al.2, the basic principle of the BChl aggregation is based on molecules properties that can serve as donors and acceptors electron. In the process of energy transfer, porphyrin aggregation and BChl in solution affect the structure of its macrocycle electronic23,24.

Fig. 3. Tookad® aggregates in acetone (A) and methanol (B) in the ratio of acetone/methanol: distilled water: 3.0 : 0.0 ( ); 2.0 :1.0 ( ); 1.0 : 2.0 ( . . . .); 0.0 : 3.0 ( )

Water is a very unique nucleophile for BChl because not only can it serve as electron donors, but it also produces hydrogen bonding23. Figure 3 shows that the addition of water lowers the absorbance of Qy1 and Qy1 also experienced peak shift towards bathochromic. This Qy1 shift formed a new absorption band which is stated by Qy2. The presence of water forming Qy2 proves that Tookad® experienced aggregation. Qy2 asymmetric absorption as indicated by the peak shift to the infrared region ~894.980 nm in acetone and ~895.029 nm in methanol indicates the competition between water molecules and the acetone/methanol to coordinate with Tookad®. Furthermore, the formation of Qy2 aggregate was also demonstrated through the Soret band shift towards hypsochromic in acetone and methanol. Leenawaty stated that the presence of polar solvents can interact with BChl molecules, such as


hydrogen bonding with Magnesium atoms on one or two sides of the axial coordination15. Katz and Gottstein et al. have studied the aggregation of chlorophyll molecules23,25. They reported that the aggregation of the chlorophyll is influenced by the interaction of the electron donor in peripheral carbonyl group at C-13 (ring V) and electron acceptor at the centre of magnesium atoms between molecules with solvent. These interactions can occur directly or if there is a presence of multifunctional ligands, such as water. This aggregation also occurs as a result of interaction between phytil-phytil as well as the interaction between tetrapyrrole -π system26,27. In this study, Qy2 shift proves that when there are hydrogen bonds in water there is interaction between the electron donor in carbonyl of group peripheral and electron acceptor on Palladium as the central atom to form Tookad® aggregates. Figure 3 shows that the spectra pattern has three main absorption bands, namely Qy, Qx, and Soret band which on excited transition in the first (S0 S1) and second singlet state (S0 S2)15,23.

Table 2. Comparison of the absorption peak shifts in Qy due to aggregation in acetone and methanol

ratio acetone methanol acetone/methanol:

distilled water Qy start

(nm) Qy end (nm) ΔQy (nm)

Qy start (nm)

Qy end (nm)

ΔQy (nm)

2.0 : 1.0 760.00 894.980 134.978 755.07 895.029 139.963 1.0 : 2.0 760.00 894.980 134.978 755.07 889.967 134.901

Bathochromic shift in the ratio of acetone/methanol : distilled water; 1.0 : 2.0 has a different response between

Tookad® were aggregated with distilled water in acetone and methanol (Figure 3A, Figure 3B and Table 2). Figure 3 and Table 2 show the axial coordination bond of Tookad® lower in methanol than acetone. The position of coordination penta in acetone and hexa coordination in methanol at the monomers form change into penta/hexa dimer coordination in the presence of water molecules. Fig. 4. The pattern of spectra changes (A1, B1) and different spectra (A2, B2) of Tookad® aggregates against irradiation in acetone (A) and

methanol (B) and its degradation products (P1 and P2) from 0 min to 60 min with time interval of every 10 min


The effect of irradiation on the Tookad® aggregates (methanol/acetone : distilled water; 2.0 : 1.0) is shown in

Figure 4. Figure 4 shows the irradiation treatment resulting in Tookad® having photobleaching. In acetone, the decrease in the absorption peak is significant at Qy1, Qy2, Qx, and Soret followed by the appearance of the absorption band of degradation products (P1 and P2). These results differ in the methanol solvent that only decreased the peak absorbance at Qy, Qx, and Soret. This fact can be seen by comparing Qy2 in Figure 4 between acetone (A) and methanol (B). Tookad® aggregates (Qy2) are significantly more susceptible to irradiation than Qy1. Research conducted by Leenawaty reported that irradiation has a correlation to the ligands position of BChl molecule10. 3.3. Tookad® aggregates-acidification responses to irradiation in acetone and methanol 3.3.1. Tookad® aggregates-lactic acid responses to irradiation

Irradiation treatment for the Tookad® aggregates with the addition of lactic acid is shown in Figure 5. Before irradiation treatment, the addition of lactic acid in the Tookad® aggregates shows that Tookad® degraded. This degradation also looked after Tookad® having been irradiated using polychromatic light.

Fig. 5. The pattern of spectra changes (A1, B1) and different spectra (A2, B2) Tookad® aggregates-lactic acid against irradiation in acetone (A)

and methanol (B) and its degradation products (P1 and P2) from 0 min to 60 min with time interval of every 10 min


In acetone, irradiation resulted in degradation at Qy1, Qy2, Soret and formed the degradation products P1 at

~429.96 nm and P2 at ~655.02 nm, whereas in methanol, Tookad® was only degraded in the Soret and Qy2 (Figure 5). As irradiation treatment in Tookad® aggregates in methanol (Figure 5B), the overall band Qy1 form aggregates Qy2 which was noticeably degraded faster than Tookad® that its Qy1 aggregated partially in acetone. These results prove that the axial ligand bonding, hexa coordination, and dimerization Tookad® aggregates-lactic acid in methanol degraded faster than with acetone. The increase in absorption peak at about ~429.96nm and ~655.29 nm in acetone (Figure 5A) is also an indication that oxidation occurs on the second ring of bacteriochlorin structure and produces structures of 3-Acetyl-chl and chlorine10. 3.3.2. Tookad® aggregates-sulphuric acid responses to irradiation

Irradiation of the Tookad® aggregates-sulfuric acid in acetone resulted in the decrease of peak absorbance at Qy1 and Qy2 band and the formation of degradation products: P1, P2, and also P3 at wavelength ~654.98 nm (Figure 6). In methanol, irradiation treatment of Tookad® aggregates-sulphuric acid only degrades the Qy and Soret band, as happened in the Tookad® aggregates-lactic acid in methanol.

Fig. 6. The pattern of spectra changes (A1, B1) and different spectra (A2, B2) Tookad® aggregates-sulphuric acid against irradiation in acetone

(A) and methanol (B) and its degradation products (P1, P2, and P3) with time interval of every 2.5 min at 0 min to 10 min and time interval of every 10 min at 10 min to 60 min


Looking at Figure 6, the differences of spectra pattern between addition of sulphuric acid and lactic acid seem to

be influenced by the type of functional groups contained in each of these acids. A study on the addition of hydrochloric acid (HCl) reported that the group-Cl is auxochrome meaning that saturated group can change the maximum absorption intensity when bound to the chromophore. Baucher dan Katz refer to the mechanism as Metalo complex formation28 At the time Tookad®starts being irradiated, pigment degradation begins with the open ring on tetrapyrrole group to form a linear tetrapyrrole oxidized. In the photodegradation process, the presence of oxygen can produce singlet oxygen and hydroxyl radicals which in turn react with tetrapyrrole to form peroxides and free radicals more. Peroxides and free radicals that arise trigger a macrocyclic damage and loss of color.

The absorbance decrease in Figure 6 shows the degradation of Tookad® molecules that followed the appearance of isosbestic points as an indication forming degradation products at each irradiation time interval. Figure 6 also shows that the Qy band degrades more than the Soret band. Degradation ratio between the Soret and Qy bands of Tookad® increased following the length of time of irradiation. Wavelength shift does not occur in the spectra during the irradiation time. This indicates that Tookad® is quite stable and did not undergo isomerization from trans to cis like in most carotenoid degradation mechanisms. The conditions of increasing the absorption peak on Figure 6 indicate degradation product, including 3-Acetyl-chl, chlorine, and its epimer. In addition, degradation process can form other products; other products such as bilin (tetrapyrrole with open ring) are experiencing a shift toward blue region10.

4. Conclusion

The results showed that Tookad® aggregated with the addition of water and degraded by the addition of acid and polychromatic light irradiation. The presence of water at a ratio of acetone/methanol: distilled water: 1.0 : 2.0 and 2.0 : 1.0 resulted in Tookad® having aggregated that is expressed through bathochromic shift of the Qy band and shifts of the Soret band. Similarly, the addition of the weak and strong acid resulted in Tookad® having degraded that is expressed by changing the position of Soret, Qx, and Qy band. Polychromatic light irradiation resulted in Tookad® having degraded the Soret band and the significant degradation of the Qy band is accompanied by the formation of degradation products as P1, P2, and P3 (Tookad® aggregates-sulphuric acid in the solvent acetone). This degradation product is indicated as 3-Acetyl-Chl at P1, chlorine in P2, and P3 as epimer Tookad® molecules. Degradation products P1, P2, and P3 need to be tested further through analysis and identification using High Performance Liquid Chromatography-3 Dimensional and Liquid Chromatography Mass Spectroscopy. Acknowledgments

Authors thank for many persons have encouraged, made suggestions, and supported us to complete this research. Authors are grateful to Prof. Hugo Scheer for his support, especially Tookad® availability. Authors give deep appreciation to Dr.Martanto Martosupono, Lia Kusmita, Katarina Purnomo, and Widhi Handayani who read portions of the manuscript and provided helpful discussions. References 1. Bonnett R, Martinez G, Photobleaching of photosensitizer used in photodynamic therapy. Tetrahedron 2001;57(591):9513–9547. 2. Luksiene Z. Photodynamic therapy:mechanism of action and ways to improve the efficiency of treatment. Medicina 2003;39(12):1137–1150. 3. MacDonald IJ, Dougherty TJ. Basic principles of photodynamic therapy. J Phorphyrins and Phthalocyanines 2001;5:105–129. 4. De Rosa MC, Crutchley RJ. Photosensitized singlet oxygen and its application. Coordination Chem Rev 2002;233–234:351–371. 5. Derycke ASL, de Witte PAM. Liposomes for photodynamic Therapy. Advanced Drug Delivery Rev 2004:56:17-30. 6. Nyman ES, Hynninen PH. Research advances in the use of tetrapyrrolic photosensitizers for photodyanmic therapy. Photochem Photobiol

2004;73:1–28. 7. Rahayu P, Limantara L. Studi lapangan kandungan klorofil in vivo beberapa spesies tumbuhan hijau di Salatiga dan sekitarnya [Field

studies in vivo chlorophyll content of some species of green plants in Salatiga and surrounding areas]. Seminar Nasional MIPA 2005,Depok. Universitas Indonesia; 2005. [ Bahasa Indonesia].

8. Koyama Y, Limantara L, Nishizawa E, Misono Y, Itoh K. Presence of penta- and hexa-coordinated state in T1 and cation-radical Bacteriochlorophyll a, and generation of cation radical by photo-excitation of the aggregated forms as revealed by transient raman and


transient absorption spectroscopies. In: Woodruff W, editor. Seventh International Conference on Time-Resolved Vibrational Spectroscopy.. Santa Fe: Proceeding Conference; 1995.

9. Vladkova R. Chlorophyll a self-assembly in polar solvent-water mixtures. Photochem Photobiol 2000;71(1):71–83. 10. Limantara L, Koehler P, Wilhelm B, Porra RJ, Scheer H. Photostability of bacteriochlorophyll a and derivatives: potential sensitizers for

photodynamic tumor therapy. Photochem Photobiol 2006;82:770–780. 11. Alsuhendra D, Muchtadi D, Sastradipradja, Wresdiyati T. Daya anti hiperkolesterolemia "Zinkofilin" (kompleks seng dengan feofitin).

[Antihypercolesterolaemic activity of zincophyllin]. Jurnal Teknologi dan Industri Pangan 2003;14(2):129–135. [Bahasa Indonesia] 12. Heriyanto, Limantara L. Fotostabilitas Zn, Cu, dan Mg bakterioklorofil a (Bchl a) sebagai sensitizer potensial dalam terapi fotodinamika:

studi dalam pelarut aseton dan metanol [Photostability of Zn-, Cu and Mg-Bacteriochlorophyll a (BChl a) as a potential sensitizer in photodynamic therapy: a study in acetone and methanol]. In: Lia K., editor. Prosiding Seminar Nasional Pigmen. Salatiga: Satya Wacana Christian University; 2007.

13. Hervé Brun P, de Groot JL, Dickson EFG, Farahani M, Pottier RH. Determination of the in vivo pharmacokinetics of Palladium-bacteriopheophorbide (WST09) in EMT6 tumor-bearing Balb/mice using graphite furnace atomic absorption spectroscopy. Photochem Photobiol 2006;3:1006–1010.

14. Vakrat-Haglili. The microenvironment effect on the generation of reactive oxygen spesies by Pd-Bacteriopheophorbide. JACS 2005;127: 6487–6497.

15. Limantara L, Sakomoto S, Koyama Y, Nagae H. Effects of nonpolar and polar solvents on the Qx and Qy energies of bacteriochlorophyll a and Bacteriopheophytin a. Photochem Photobiol 1997;65:330–337.

16. Misono Y, Nishizawa E, Limantara L, Koyama Y, Itoh K. Solvent effects on the resonance raman spectra of Bacteriochlorophyll a cation radical. Chem Phys Lett 1995;236:413–418.

17. Limantara L, Fujii R, Koyama Y et al. Gene-ration of triplet and cation radical Bacteriochlorophyll a in Carotenoidless LH1 and LH2 antenna complexes from Rhodobacter sphaeroides. Biochem 1998;37:17469–17486.

18. Nishizawa EI, Limantara L, Nanjou N, Nagae H, Kakuno T, Koyama Y. Solvent effects on triplet-state Bacteriochlorophyll a as detected by transient raman spectroscopy and the environment of Bacteriochlorophyll a in the light-harvesting complex of Rhodobacter sphaeroides R26. Photochem Photobiol 1994;59(2):229–236.

19. Fiedor J, Fiedor L, Kammhuber N, Scherz A, Scheer H. Photodynamics of the Bacteriochlorophyll-Carotenoid system 2: influence of central metal, solvent and β-Carotene on photobleaching of Bacteriochlorophyll derivatives. Photochem Photobiol 2002;76(2):145–152.

20. Jeffrey SW, Mantoura RFC, Wright SW. Phytoplankton pigments in oceanography: guidelines to modern method. UNESCO Publishing, Paris Kephart, J.C. Escon. Bot., 2003:9:3–38.

21. Watanabe T, Kobayashi M. Electrochemistry of chlorophylls. In: Scheer H, editor. Chlorophylls. Boca Raton: CRC Press; 1991. p. 287–315. 22. Katz JJ, Norris JR, Shipman LL, Thurnauer MC, Wasielewski MR. Chlorophyll function in the photosyntetic reaction center. Ann Rev

Biophys Bioeng 1978;7:393–434. 23. Katz JJ, Shipman LL, Cotton TM, Janson TR. Chlorophyll aggregation. In: Doplin D, editor. The porphyrin: physical chemistry part C. New

York: Acad. Press; 1978;5. 24. Scherz A, Rosenbach-Belkin V, Michalski TJ, Worcester DL. Chlorophyll aggregates in aqueous solutions. In: Scheer H, editor.

Chlorophyll. Boca Raton: CRC Press; 1991.p.237–268. 25. Gottstein J, Scherz A, Scheer H. Bacteriochlorophyll aggregates in positively charged micelles. Biochimica et Biophysica Acta 1993;

1183:413–416. 26. Oba T, Watanabe T, Mimuro M, Kobayashi M, Yoshida S. Aggregation of chlorophyll a in aqueous methanol. Photochem Photobiol

1996;63(5):639–648. 27. Sasaki S, Omoda M, Tamiaki H. Effects of C8-substituents on spectroscopic and self-aggregation properties of synthetic

Bacteriochlorophyll-d analogues. Photochem Photobiol 2003;162:307–315. 28. Baucher LJ, Katz JJ. Aggregation of metallochlorophylls. J Am Chem Soc 1967;89:4703–4708.

the photosensitizer stabilities of tookad® on aggregation, acidification, and day-light irradiation

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