Magnesium octa[(4’-benzo-15-crown-5)oxy]phthalocyanine in phosphate buffer: supramolecular organization, cytotoxicity and accumulation/localization in tumor cells of HeLa Текст научной статьи по специальности «Фундаментальная медицина»

Phthalocyanines Фталоцианины

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Paper Статья

DOI: 10.6060/mhc180899l

Magnesium Octa[(4r-benzo-15-crown-5)oxy]phthalocyanine in Phosphate Buffer: Supramolecular Organization, Cytotoxicity and Accumulation/Localization in Tumor Cells of HeLa

M. A. Lapshina,ae@1 S. I. Norko,b V. E. Baulin,cd A. A. Terentiev,abe A. Yu. Tsivadze, N. F. Goldshlegera@2

aInstitute of Problems of Chemical Physics RAS, 142432 Chernogolovka, Russia bLomonosov Moscow State University, 119991 Moscow, Russia

cFrumkin Institute of Physical Chemistry and Electrochemistry RAS, 119071 Moscow, Russia dInstitute of Physiologically Active Compounds RAS, 142432 Chernogolovka, Russia Moscow Region State University, 105005 Moscow, Russia @1 Corresponding author E-mail: lapshina@icp.ac.ru @2Corresponding author E-mail: nfgold@icp.ac.ru

For the first time, magnesium octa[(4'-benzo-15-crown-5)oxy]phthalocyanine (Mgcr8Pc) was explored as a potential PDT agent. Presence of crown-containing fragments in the periphery of the tetrapyrrole macrocycle makes the compound soluble in water. Solutions of Mgcr8Pc in water and in phosphate buffer (PBS) are stable for a long time. Modification of Mgcr8Pc solutions in PBS with sodium deoxycholate led to partial monomerization of Mgcr8Pc and hence to formation of a fluorescent species. The cytotoxicity ofMgcr8Pc was determined with respect to HeLa cells and its accumulation/localization in the cells was studied by fluorescence microscopy. The increase in signal intensity indicates the accumulation of Mgcr8Pc in HeLa cells over time. The cell morphology was found to remain practically intact when subjected to Mgcr8Pc at concentration of 5.00 yM for 24 h. The results obtained allow us to continue the study of this interesting class of compounds.

Keywords: Phthalocyanine, crown ether conjugates, solubility in water, spectral properties, supramolecular organization, sodium deoxycholate, HeLa cells, cytotoxicity, accumulation, localization, fluorescence microscopy.

Окта[(4'-бензо-15-краун-5)окси]фталоцианинат магния в фосфатном буфере: супрамолекулярная организация, цитоксичность, накопление и локализация в опухолевых клетках HeLa

М. А. Лапшина,^1 С. И. Норкой В. Е. Баулин,^ А. А. Терентьев,^ А. Ю. Цивадзе^ Н. Ф. Гольдшлегер^2

aИнститут проблем химической физики РАН, 142432 Черноголовка, Россия bМосковский государственный университет имени М.В. Ломоносова, 119991 Москва, Россия Институт физической химии и электрохимии им. А.Н. Фрумкина РАН, 11999 Москва, Россия Институт физиологически активных веществ РАН, 142432 Черноголовка, Россия eМосковский государственный областной университет, 105005 Москва, Россия

E-mail: lapshina@icp.ac.ru @2E-mail: nfgold@icp.ac.ru

Окта[(4'-бензо-15-краун-5)окси]фталоцианинат магния (Mgcr^c) в фосфатном буфере впервые исследуется как потенциальный ФДТ-агент. Присутствие краун-содержащих фрагментов на периферии тетрапиррольно-го макроцикла обеспечивает растворимость соединения в водной среде. Растворы MgcrPc в воде и фосфат-

ном буфере стабильны в течение длительного времени в обычных условиях. Введение дезоксихолата натрия приводило к частичной мономеризации и формированию флуоресцентно-активных частиц, соответственно. Определена цитотоксичность Mgcr8Pc и показано его накопление и локализация в клетках HeLa. Согласно флуоресцентной микроскопии, морфология клеток не изменяется после 24-х часового действия Mgcr8pc в дозе 5.00 мкМ.

Ключевые слова: Фталоцианин, конъюгаты с краун-эфирами, растворимость в воде, спектральные свойства, супрамолекулярная организация, дезоксихолат натрия, опухолевые клетки HeLa, цитотоксичность, накопление, локализация, флуоресцентная микроскопия.

Introduction

Phthalocyanines (Pc) and their supramolecular aggregates are applied in molecular electronics, chemical sensors, catalysis, biology, and medicine, including photodynamic therapy (PDT).[1-3] PDT, which is an actively developing field of research for the treatment of a variety of cancers, is based on the capability of photosensitizer (PS) to accumulate preferentially in the tumor and by a precise illumination to form highly reactive oxygen species, which result in cytotoxic reactions in the cells.[4]

Along with chemical stability, phthalocyanines exhibit absorption bands with intense n-n* transitions in the range of 650-850 nm (e > 105 M-1cm-1), low toxicity, and high quantum yield of the triplet state, which makes Pc a feasible PS in fluorescent diagnostics and PDT of malignant neo-plasms.[5-8] A comparison of some Pcs as photosensitizers has already been made in vitro (e.g. see[9] and references therein). Complexes containing Zn2+, Al3+, Ga3+, Mg2+, etc. in the centre of the macrocycle seem to be most suitable for PDT. For instance, hydroxyaluminum phthalocyanine trisulfonate (Photosens)[7] has been approved for clinical PDT in Russia. In order to generate reactive oxygen species (largely singlet oxygen), Pc is to be in its monomer state in solution. However, this is difficult to achieve in view of (a) aggregation of Pc molecules in polar media and (b) poor solubility of phthalocyanines in water.

In this work, magnesium octa[(4'-benzo-15-crown-5)-oxy]phthalocyanine (Mgcr8Pc, Figure 1) is explored as a potential PDT agent. The study includes (a) the determination of cytotoxicity of Mgcr8Pc with respect to HeLa cells

and (b) its accumulation/localization in tumor cells HeLa in vitro. Considerable attention is also paid to the state of Mgcr8Pc in the cell growth medium as well as supramolecular organization of Pc in microheterogeneous media based on anionic surfactants, including biocompatible sodium deoxycholate, in phosphate buffer (p^ 7.4). The presence of the crown fragments improves the solubility of complexes and promotes the self-assembling of Pc with a guest cation,[10-12] as well as opens up a way to preparation of materials with remarkable physicochemical characteris-tics.[13-14]

Experimental

General

The synthesis of magnesium octa[(4'-benzo-15-crown-5) oxy]phthalocyanine (Mgcr8Pc) was carried out as described elsewhere.[15] Commercially available sodium dodecyl sulphate (SDS), sodium deoxycholate (SDC) (Aldrich, 98 %) and NaCl (extra-pure grade) were used without additional purification. Salts used in preparation of phosphate buffer saline with pH 7.4 (hereinafter PBS) also were of extra pure grade; according to protocol, the NaCl, KCl, Na2HPO4, and KH2PO4 concentrations were 137, 2.7, 10 and 1.76 mM, respectively. Solutions were prepared by using double distilled or deionized water. The concentration of the Mgcr8Pc stock solution was equal to 594 ^M. All solutions were stored in the dark.

To study the effect of salts, equal number of mL of the Mgcr8Pc/SDC/NaCl solution ([Mgcr8Pc]=9.3-10"6 M, [SDC]=0.0175 M, [NaCl]=0.139 M) was placed in two identical cuvettes. In one of them we added a dry sample of KCl: total

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concentration of salts-(NaCl + KCl) in solution under study was equal to 0.16 M. The NaCl sample was also introduced in second cuvette. In both cases, the ionic strength of the solution and the concentrations of Mgcr8Pc and SDC did not change. The absorption spectra were recorded in time.

Determination of Mgcr8Pc Absorbed

HeLa (human cervical adenocarcinoma) cells were incubated in Eagle MEM medium (EMEM, hereinafter merely "medium") modified by the addition of Mgcr8Pc solution in PBS. In 24 h, the solution was poured out and the cells were washed several times with 2 mL portions of PBS. The absorption spectra of the collected Mgcr8Pc were close to those of Pc aggregates and Pc in reference to Mgcr8Pc/medium/PBS solutions. The concentration of Mgcr8Pc was determined spectrophotometrically. The amount of Mgcr8Pc absorbed by the cells ([Mgcr8Pc]ab) was calculated from the difference between the initial concentration of Pc ([Mgcr8Pc]0) and the amount of Pc collected after the incubation stage ([Mgc ^Pcfr):

[Mgc^Pc] 0, yM [Mgc^Pcf yM [Mgcr8Pc]ab, % 20 14 ~30

10

6.08

~40

For [Mgcr8Pc]<5 yM, washing solutions contained practically no Pc.

Optical absorption spectra were recorded with a Specord M-40 spectrophotometer by using 1-, 2-, and 10-mm quartz cells. In some cases, the spectra were deconvoluted into components.

Fluorescence spectra of Mgcr8Pc/SDC in PBS solutions stored for 24 h were recorded with a PerkinElmer LS55 spec-trofluorimeter at room temperature. The slit width was 10 nm; the wavelength of excitation was 614 nm.

Biological Experiments

Cell culture. HeLa cells were obtained from the Russian collection of cell cultures of vertebrates. The tumor cells were grown in EMEM medium (PanEco, Russia) containing 10 % embrionic whey (Biowest, France), penicillin (50 units/mL), and streptomycin (50 mg/ml) at 37 °C in a 5 % CO2 atmosphere.

MTT test. The influence of Mgcr8Pc on cell growth and viability was studied upon their staining with 3-(4,5-dime-thyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich).[16-18] The cells were dissipated in 96-well plates in the amount of 5T03 cells/100 yL for each well. In order to determine the cytotoxity of Mgcr8Pc, its solution in PBS (0.5-25 yM) was added to the incubation medium. The incubation period in the presence of Pc was 24 h. Dark toxicity of Mgcr8Pc towards HeLa cells was studied both with and without rinsing the samples with PBS. In the first case, the cells were washed twice with 50 yL of buffer PBS before applying MTT dye. After this, 0.5 mg/mL MTT was added and the plate was held at 37 °C in a 5 % CO2 atmosphere for 3 h. Then the incubation medium was removed and the formed formazan crystals were dissolved in DMSO for 10 min. The MTT assay was established at 570 nm (Tecan SPARK 10M device). Cells' viability was estimated as the percentage of MTT stained control cells. The IC50 dose (concentration of a compound that reduces MTT staining by 50 %) was found using the median effect analysis. Each experiment was repeated three times.

Fluorescence Microscopy. The cells grown on cover glasses (450 000 in each) were incubated with Mgcr8Pc added in concentrations of 5 yM for 3 or 24 h, respectively. After incubation, the remaining Mgcr8Pc was washed out with PBS. Then the cells were held in 4 % paraformaldehyde solution in PBS at r.t. for 30 min, in 0.5 % solution of Triton X-100 for 10 min, stained

in DAPI (4',6-diamidino-2-phenylindole-2HCl) solution for 10 min, washed with deionized water and dried at r.t. in the dark. After this, the samples were placed in a drop of fixing fluid Fluo-romount (Sigma-Aldrich, Germany) on a cover glass. In the same way, HeLa cells were also treated with Mgcr8Pc without fixing. In this case, the cells grown on cover glasses were placed in a drop of PBS solution just before viewing. Accumulation/localization of Mgcr8Pc in fixed and non-fixed tumor cells was explored with an Axio Scope.A1 fluorescence microscope (Carl Zeiss, Germany) using A-Plan 40x/0.65 Ph2, A-Plan 40x/0.65 M27, N-Achroplan 100x/1.25 Oil M27, and N-Achroplan 100x/1.25 Oil Ph3 M27 objectives and a set of fluorescence filters: Fs 49 DAPI (EX G 365, EM BP 445/50), Fs 45 HQ TexasRed (EX BP 560/40, and EM BP 630/75 - in combination with a high-resolution digital camera AxioCam MRc 5 and Zen 2012 software (blue edition).

Results and Discussion

In dichloromethane, the spectra of Mgcr8Pc (Figure 1) typically exhibit a strong n-n* transition at longer wavelengths (e >105 M-1cm-1) and a weaker one at shorter wavelengths (0-band), and also a weak but broad B-band (also called a Soret band) in the UV range (Figure 2a). Such spectral features are typical to monomeric metal phthalo-cyanine complexes.

Absorption Spectra of Mgcr8Pc in Various Media

Absorption spectra of Mgcr8Pc in water, in PBS and in EMEM medium are similar. As an example, Figure 2a presents the absorption spectrum of Mgcr8Pc in PBS (curve 2). In contrast to dichloromethane (curve 1), the 0-band of Mgcr8Pc in aqueous medium is blue-shifted and strongly broadened, which is indicative of the aggregation of phthalocyanine molecules. The blue-shift is caused by the n-n stacking that is characteristic of dimers, associates and other aggregates.[11] As follows from Figure 2b, the spectrum of Mgcr8Pc in EMEM medium which is similar to that in PBS can be readily deconvoluted into four Gaussian functions. The latter is considered by us only as an indication of the possible variety of particles forming in the solution.

As it is seen in Figure 3a, the addition of anionic SDC to Mgcr8Pc/PBS solution ([SDC] > cmc) causes a red shift of short-wavelength B-band and also the narrowing and intensification of the 0-band. In the case of Mgcr8Pc/ SDC/NaCl (Figure 3a, spectrum 3 and see also[19]), the shape of absorption spectrum and a higher e value for the 0-band testify a higher concentration of Pc monomer as compared to Mgcr8Pc/SDC/PBS solution (Figure 3 a, spectrum 2), and the NaCl concentration is close to the physiological value in both systems. It should be also noted that the spectral characteristics such as the shape of spectrum and extinction coefficient (Figure 3a, spectrum 2) remained almost unchanged after the subsequent NaCl addition to the Mgcr8Pc/SDC/PBS solution.

The effect of the Na+ and K+ ions on the spectral characteristics of the micro-heterogeneous system based on Mgcr8Pc/SDC is also shown in Figure 3b. It is seen that the KCl addition leads to absorption spectrum (Figure 3b, spectrum 2) close to that of the Mgcr8Pc/SDC/PBS system (Figure 3a, spectrum 2).

Figure 2. (a) Absorption spectra of Mgcr8Pc in dichloromethane

(curve 1) and in phosphate buffer (curve 2); (b) Fragment

of the Mgcr8Pc spectrum in EMEM medium and its deconvolution

into Gaussian functions; the red line is the envelope

of the deconvolution contours, and the thin black solid lines are

Gaussian contours.

The absorption spectra of Mgcr8Pc in SDS/water and SDS/PBS are also markedly different ([SDS] > cmc in both systems): Pc as monomer in the former case (Figure 4, spectrum 1 and see also[15]), no clearly pronounced maximum, a larger half-width of the g-band, and lower e value in the latter case (Figure 4, spectrum 2).

Thus, the present in PBS of the K+ ions (see Experimental) whose diameter (2.66 Â) significantly exceeds the diameter of the cavity in 15-crown-5 (1.7-2.2 Â),[1011] facilitates the cation-induced aggregation of Mgcr8Pc even in the presence of anionic surfactants such as sodium deoxycholate and sodium dodecyl sulfate. In all cases, spectral features obtained appear in the presence of the K+ ions (see Figures 3 and 4). It was previously shown[20] that the K+ ions in the KCl aqueous solution lead to the formation of stable charged forms (dimers) of octa-crown metal phthalocyanines.

Photoexcitation of ' Mgcrpc/SDC/PBS Solutions

Figure 3. (a) Absorption spectra of Mgcr8Pc/PBS,

Mgcr8Pc/SDC/PBS and Mgcr8Pc/SDC/NaCl solutions

(1, 2 and 3, respectively); [Mgcr8Pc]=9.3-10-6 M,

[SDC]=0.016 M. (b) Salt effect on absorption spectra of Mgcr8Pc

in micro-heterogeneous systems:

SDC/NaCl ([NaCl]=0.139 M + 0.025 M; spectrum 1)

and SDC/NaCl/KCl ([NaCl]=0.139 M and [KCl]=0.022 M;

spectrum 2). Insert: first derivatives of spectral bands 1 and 2,

respectively.

Figure 5a presents the normalized fluorescence and excitation spectra for Mgcr8Pc/PBS/SDC solution. The excitation spectrum in Figure 5a (curve 2) corresponds

Figure 4. Absorption spectra of Mgcr8Pc in SDS/water (curve 1) and SDS/PBS (curve 2) solutions. Insert: first derivatives of spectral bands 1 and 2, respectively.

60-

Figure 5. Mgcr8Pc/SDC/PBS solution: (a) fluorescence spectrum as excited at Xex=614 nm (curve 1) and excitation spectrum as taken at Xem=704 nm (curve 2); (b) relative fluorescence intensities for Mgcr8Pc/SDC/NaCl (curve 1) and Mgcr8Pc/SDC/PBS (curve 2) systems.

to the absorption spectra of the Mgcr8Pc monomer in dichlo-romethane (Figure 2a, curve 1) or in the SDS/H2O medium at [SDS] > cmc (critical micelle concentration), but strongly differs from the absorption spectrum of Mgcr8Pc in PBS/SDC solution ([SDC] > cmc]) (see Figure 4, curve 1 and Figure 3, curve 2, respectively). This behavior is also characteristic of the Mgcr8Pc in the SDS/PBS microheterogeneous system.

Relative fluorescence intensities for Mgcr8Pc/PBS/ SDC and Mgcr8Pc/NaCl/SDC solutions are shown in Figure 5b. It is seen that the fluorescence intensity of Mgcr8Pc/ NaCl/SDC is stronger than that of Mgcr8Pc/PBS/SDC. This may be due to a higher degree of the Mgcr8Pc aggregation in the PBS/SDC medium (see also Figure 3).

Thus, the observation of the Mgcr8Pc fluorescence in the microheterogeneous PBS/SDC and PBS/SDS systems confirms (i) the presence of a certain concentration of monomeric Pc and (ii) the absence of fluorescence quenching as a result of screening Mgcr8Pc molecules from the aqueous medium by SDC aggregates (micelles) or SDS micelles, since the fluorescence of H-dimers and larger Pc aggregates is reportedly quenched.[21]

At normal conditions, Mgcr8Pc solutions in water and phosphate buffer remain stable for a long time. When Mgcr8Pc/SDC/PBS samples were subjected to diffuse illumination for a long time, the optical density of the Band 0-bands in the absorption spectrum decreased smoothly, which was accompanied by the appearance of new bands at shorter wavelengths, just as in case of Mgcr8Pc/SDC/ NaCl (see Figure 6). In other words, we observe the true photobleaching of Pc solution typical to Pc monomers. According to,[22] the photodestruction of Pc yields respective phthalimides, as shown also for Pc with four annulated crown fragments in the NMR spectra[23] and for Pc with eight methyl phosphonate groups.[24]

Cytotoxicity of Mgcr^Pc and Its Accumulation/ Localization in HeLa Cells

Metal phthalocyanines containing crown ether solubilize in microheterogeneous SDC-based media largely in the form of monomers in the presence of physiological concentrations of sodium chloride (see Figure 3 and also[19]),

but our control experiments and literature data have revealed that SDC is quite toxic not only for HeLa but also for some other cells.[25-27] This circumstance impeded a correct exploration of the cytotoxicity of the fluorescing Mgcr8Pc/SDC/ PBS system. 8

At this stage, we investigated as a result the toxicity of Mgcr8Pc itself and also its ability to penetrate and to accumulate in HeLa cells.

The state of the Mgcr8Pc in the cells and its possible changes in comparison with Pc in EMEM medium (see above) are of current interest. Based on the known data on the supramolecular organization of Mgcr8Pc in microhet-erogeneous media[28] and due to non-covalent interactions of various types (see work[29] and references therein), certain modifications of the Pc state in cells could well be expected.

Cytotoxicity of Mgcr^Pc

Figure 7 shows the results of MTT tests of cytotoxicity of Mgcr8Pc toward tumor cells HeLa. The data obtained

Figure 6. Photobleaching of Mgcr8Pc in microheterogeneous system SDC/NaCl, Xex=578 nm. Inserts: (a) A683 vs. time; (b) first and second derivatives of the initial Mgcr8Pc spectrum (before irradiation). [Mgcr8Pc]=7.4-10-6 M, [SDC]=0.016 M, [NaCl]=0.1016 M.

allow us to state that the cell proliferation is supressed in a dose-dependent mode.

It should be also noted that the washing of cells with PBS or its absence before dye applying do not have a determining influence on the staining MTT value determined: the IC50 concentrations were found to have a value of 8.48 and 9.58 ^M, respectively.

Accumulation of Mgcr^c in HeLa Cells

The intracellular content and distribution of the test compound was evaluated by fluorescence microscopy. Fig-

Figure 7. Cytotoxicity of Mgcr8Pc vs. tumor cells HeLa; before applying the MTT dye the sample was washed with PBS.

ure 8 illustrates the Mgcr8Pc accumulation and localization in HeLa cells. The phase contrast images show (Figure 8-2A) that as a result of the incubation with [Mgcr8Pc]=5 |M for 3 h the cell morphology remained practically unchanged. The distribution of Pc over the cells is non-uniform as evidenced by brightly colored granules in Figure 8-2C.[30] The incubation of cells with Mgcr8Pc for 24 h noticeably increases the signal from Mgcr8Pc in the cytoplasm of cells (Figure 8-3C).

The results on the accumulation of Mgcr8Pc in non-fixed HeLa cells also confirm that Mgcr8Pc absorbs in cells at [Mgcr8Pc]=5 |M for 24 h (Figure 9) and localizes predominantly in the cytoplasm of the latter.

Thus, Mgcr8Pc is soluble in water, PBS, and culture medium EMEM, where it is present mainly in an aggregated state. Addition of sodium deoxycholate promotes the partial Pc monomerization and appearance of fluorescent species, respectively. Treatment of HeLa cells with Mgcr8Pc/ PBS solution in culture medium led to the penetration/ accumulation of Pc in the cells. Toxicity of Mgcr8Pc (8.48 ^M) toward HeLa is higher as compared to that of some substituted Pc of Mg, Zn, metal-free Pc and hydroxyaluminum trisulfophthalocyanine (Photosens, Russia). So, the latter adsorbed on polymer nanoparticles shows low cytotoxicity toward HeLa cells for [Pc] < 11.2 |g/mL (12.74 |M).[31] For zinc 9,10,16,17,23,24-hexa(4'-tert-butylphenoxy)-2-[2'-(4"-carboxyphenyl)ethynyl]phthalocyanine and HeLa cells, IC50 equals to 21.44 |M;[32] for other derivatives of MgPcs, ZnPcs, and 2HPcs, IC50 > 100 |M (see[3334]).

The fluorescence of Mgcr8Pc molecules located in the fixed and non-fixed cells HeLa indicates that Pc is present in them in the state really strongly differing from that in culture medium. The micrographs of fluorescence

Figure 8. Localization of Mgcr8Pc in HeLa cells. Lines: 1A-D unprocessed cells, 2A-D cells processed with [Mgcr8Pc]=5 |M for 3 h, 3A-D cells processed with [Mgcr8Pc]=5 |M for 24 h. Columns: A1-3 phase contrast, B1-3 nuclei stained with DAPI, C1-3 fluorescence of Mgcr8Pc in the cells, D1-3 merger. Scale bar 10 |m.

Figure 9. Localization of Mgcr8Pc in HeLa cells (without fixation). Lines: 1A-C unprocessed cells, 2A-C cells processed

with [Mgcr8Pc]=5 |M for 24 h. Columns: A1-2 phase contrast, B1-2 accumulation/localization of Mgcr8Pc in the cells, C1-3 merger.

Scale bar 10 |m.

microscopy evidence the accumulation and the localization of Mgcr8Pc in tumor cells of HeLa. High toxicity of Mgcr8Pc may be associated with the disruption of the cellular ion balance due to the presence of eight crown ether groups in the molecule. For instance, a recent review was devoted to crown ethers and some compounds based on them as prospective antitumor agents.[35]

Conclusions

For the first time, magnesium octa[(4'-benzo-15-crown-5)oxy]phthalocyanine was explored as a potential PDT agent. The results on the HeLa cells demonstrate that Mgcr8Pc accumulates in their intracellular space. Absorption spectroscopy data on the Pc uptake by cells are consistent with the results of fluorescence microscopy. Further studies, including the determination of the Mgcr8Pc photo-cytotoxicity, etc., are in progress.

Acknowledgments. This work was performed in accordance with the state task, state registration: 0089-20-140036, 0089-20-14-0040, and 0090-20-17-0024, and Russian Academy of Sciences (program no. 34). We are also grateful to VYu. Gak for taking the fluorescence spectra.

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30. Treatment of Mgcr8Pc with a 4 % paraformaldehyde solution in PBS or Triton X100 at their concentrations and ratios used in the fixation and permeabilization steps did not result in spectral changes indicating the monomerization of Pc.

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33. [1,4,8,11,15,18,22,25-Octakis[(2-(triethylammonio)ethyl)-sulfanyl]phthalocyaninato]magnesium(II) octaiodide; [1,4,8, 11,15,18,22,25-octakis[(2-(triethylammonio)ethyl)sulfa-nyl]phthalocyaninato]zinc(II) octaiodide; [1,4,8,11,15,18, 22,25-octakis[(2-(triethylammonio)ethyl)sulfanyl]phthalo-cyanine octaiodide (MgPcs, ZnPcs and 2HPcs, respectively).

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Received 26.09.2018 Accepted 25.11.2018