Novel Zn(II) phthalocyanine with tyrosine moieties for photodynamic therapy: Synthesis and comparative study of light-associated properties

Polyhedron 162 (2019) 121–128

Contents lists available at ScienceDirect

Polyhedron journal homepage: www.elsevier.com/locate/poly

Novel Zn(II) phthalocyanine with tyrosine moieties for photodynamic therapy: Synthesis and comparative study of light-associated properties Meliha Aliosman a, Ivan Angelov a, Yavor Mitrev a, Ivan Iliev b, Mahmut Durmusß c, Vanya Mantareva a,⇑ a

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev, Bld. 9, 1113 Sofia, Bulgaria Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl. 25, 1113 Sofia, Bulgaria c Gebze Technical University, Department of Chemistry, PO Box 141, Gebze, Kocaeli 41400, Turkey b

a r t i c l e

i n f o

Article history: Received 8 November 2018 Accepted 12 January 2019 Available online 23 January 2019 Keywords: Zn(II) phthalocyanine Tyrosine Singlet oxygen Breast cancer cells Photodynamic therapy

a b s t r a c t Octa-(4-tyrosylamido) phenyl substituted Zn(II) phthalocyanine (ZnPcTyr8) was synthesized and characterized as potential photosensitizer for photodynamic therapy (PDT). The new ZnPcTyr8 was evaluated in comparison to recently synthesized tetra-(4-tyrosylamido) phenyl substituted Zn(II) phthalocyanine (ZnPcTyr) and used as a standard unsubstituted Zn(II) phthalocyanine (ZnPc). The photophysical properties of absorption and fluorescence, and photochemical properties of singlet oxygen generation and photostability as well as in vitro photocytotoxicity property were investigated. The irradiation from a light-emitting diode (LED 665 nm) with a dose of 50 J cm 2 and a fluence of 60 mW cm 2 was applied. The obtained results suggested improved photophysicochemical properties of singlet oxygen generation for tyrosine-conjugated Zn(II)-phthalocyanines. In addition the tyrosine substitution to ZnPc is liable for gentle photocytotoxicity which is in contrast to the harsh phototoxic effect determined for ZnPc on tumor and normal cell lines. Among both tyrosine substituted Zn(II) phthalocyanines, a novel ZnPcTyr8 has advantages of water solubility and proper values of the main photophysicochemical properties responsible for PDT efficacy. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction The routine of photodynamic therapy (PDT) characterizes with a systematic application of a photosensitizer (PS) which after accumulation in the target spot is illuminated with specific light within the spectra of the incubated PS [1,2]. The molecular oxygen in the vicinity of target area is of importance for optimal potential of the photosensitization reaction [3]. The adequate PS concentration and the dose of light exposure should be estimated so that the disordered cells to experience photocytotoxic effect without affecting the surrounding normal cells. Moreover the uptake and localization of a PS emerge the limitation of an undesirable photocytotoxicity. A number of drug-delivery strategies for selective transport and release are recently summarized [4]. Therefore, the progress in development of more effective target-specific PS compounds is still under spotlight of research studies [5,6]. Among the new generation heterocyclic photosensitizers with long wavelength of absorbance (>670 nm) are phthalocyanine dyes [7–10]. The skeleton of Pc includes four unsaturated rings connected with nitrogen atoms, which results in high hydrophobicity of Pcs [7]. The hydrophobic nature predicts the non-specific ⇑ Corresponding author. E-mail address: [email protected] (V. Mantareva). https://doi.org/10.1016/j.poly.2019.01.029 0277-5387/Ó 2019 Elsevier Ltd. All rights reserved.

binding to many cells and undesirable interactions with lipoproteins and surrounding biomolecules. The enhancement of solubility was the initial reason for synthesis of conjugates phthalocyanines with amino acids as substituents [7,8]. Presently, there have not enough research expertise about efficacy of the amino acidsconjugates Pcs for PDT [9,10]. The non-essential amino acids and the short peptides based on them attract the research interest because of their excellent cellular penetration and the specificity to bind selectively the pathogenic cells [11,12]. Moreover the proper conjugation can lead to synergistic action of both biologically active molecules. The physiology of the development of malignant cells involves the action of proteases which suggests that there is a connection between tumor growth and the amount of proteolyses enzymes [13]. An important characteristic of the cell creation and the apoptosis is the matrix proteases [14]. Referring to nonspecificity of the photodynamic action only the PS in close interaction with the target cells appears the critical amount PS that can limit the photocytotoxic effect. The local irradiation of the target area also aim to induce selective photocytotoxic effect [15]. The tyrosine is a natural non-essential amino acid which is known to be synthesized from phenylalanine and further it is involved in production of basic proteins in the living organisms. The capability of tyrosine to enter into central nervous system with the crossing blood–brain barrier can be useful for PDT [15]. Our

122

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

recent studies of tetra-(4-tyrosylamido) phenyl substituted zinc(II) phthalocyanine (ZnPcTyr) suggested a high efficiency of new photosensitizer the photophysical and photochemical properties [16,17]. Most of amino acids substituted phthalocyanines tend to possess the increasing solubility in polar media with minimal aggregation which can tend to improve PDT process [18]. The present study aims synthesis of Zn(II) phthalocyanine substituted with eight tyrosine units, namely 2,3,9,10,16,17,23,24-octa (4-tyrosylamido) phenyl – substituted zinc(II) phthalocyanine (ZnPcTyr8). The photophysical and photochemical properties associated with PDT efficacy of the new ZnPcTyr8 were investigated in comparison to tetra(4-tyrosylamido) phenyl- substituted Zn(II) phthalocyanine (ZnPcTyr) and non-substituted Zn(II) phthalocyanine (ZnPc). Photocytotoxicity of these three phthalocyanines was evaluated on two breast cancer cell lines (MDA and MCF-7) and a normal cell line (MCF-10) in comparative in vitro studies. 2. Experimental

in an ice bath to activate the carboxylic group of the amino acid. Then 65 mg (0.045 mmol) of octa-amino substituted ZnPc (2b) was added to the reaction mixture. The apparatus was flushed with argon and the stirring was continued at room temperature for 48 h. Then the reaction mixture was extracted with DCM. The solvent was evaporated and the obtained crude product was washed several times with hexane and iso-propylether, and purified with column chromatography with eluent DCM/EtOH (10:1). Yield: 45 %. 1 H NMR (d6-DMSO), (d: ppm): 10,08 (s, 4H), 8.94 (s, 4H), 7.66 (s, 12H), 7.01–7.30 (m, 36H, CH arom), 6.76–6.87 (m, 24H, CH arom), 4.39 (br, 8H, 4  CH, CH aliphatic), 3.05 (br, 8H, 4  CH2, CH aliphatic), 2.99 (br, 8H, 4  CH2, CH aliphatic), 1.27–1.38 (br, 144H, t-Bu). UV–Vis (DMSO): kmax (log e, dm3 mol 1 cm 1): 357 (3.97), 614 (3.59), 680 (4.31). IR [(KBr pellet) mmax (cm 1)]: 3401, 3306 (NH), 3031 (Aromatic CH), 2977, 2873 (Aliphatic CH), 1709, 1610 (NH), 1507, 1366, 1205, 1163, 1088, 1029, 896. Anal. Calc. for C224H256N24O40Zn: C, 67.43; H, 6.47; N, 8.43; Found: C, 67.42; H, 6.46; N, 8.42%. MALDI-TOF (m/z) Calcd.: 3990; Found 3671.82 [M-BocTyr (tBu)+2H]+.

2.1. Materials and apparatus The used solid chemicals and solvents were of reagent-grade quality obtained from different commercial suppliers. All solvents used for synthesis such as dimethylformamide (DMF), 1-pentanol were dried or distilled and stored over molecular sieves (4 Å). Zinc acetate dihydrate was dried before used. Sodium sulfide nonahydrate, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 4-(4,6dimethoxy[1,3,5]triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), 4-methylmorpholine (NMM), L-(tert-butoxycarbonyl)O-tert-butyl-L-tyrosine (L-Boc-Tyr(tBu)-OH) and trifluoroacetic acid (TFA) were used as received. The purity of the products was tested in each step using thin layer chromatography (TLC). Silica gel 60 Å was purchased from Merck. All reactions were carried out under argon atmosphere. 1 H NMR spectra were recorded on a BRUKER 600 MHz spectrometer using d6-DMSO as deuterated solvent. FT-IR spectra were obtained using a BIORAD SPC-3200 FTS7 spectrometer. Mass spectra were recorded using Bruker Autoflex III MALDI-TOF spectrometer using as a MALDI matrix Dithranol (DIT), trans-2-[3-(4-tertButylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB). The absorption and emission measurements were performed on a UV–Vis Jasco spectrophotometer VA570, fiber optics specifically elaborated spectrophotometer on the basis of Ocean Optics QE 65,000 spectrophotometer with Spectra Suite Software and PerkinElmer Luminescence Spectrometer LS55. Fluorescence lifetimes were studied on a time correlated single photon counting (TCSPC) method using FLUOROLOG-3 fluorometer (Horiba Jobin Yvon, Edison, NJ) equipped with a NanoLED and a standard air cooler (R928 PMT detector). The equipment has a computer system with software configured for this measurement. 2.2. Synthesis The synthetic scheme includes as starting compounds 4,5dichloro-1,2-dicyanobenzene, 4,5-bis(4-nitrophenoxy) phthalonitrile, 2,3,9,10,16,17,23,24(4-nitrophenoxy) zinc(II) phthalocyanine and 2,3,9,10,16,17,23,24(4-aminophenoxy) zinc(II) phthalocyanine. They were synthesized and purified by following the known procedures [19,20]. 2.2.1. 2,3,9,10,16,17,23,24-Octa[4-Boc-tyrosyl(tBu)amido]phenyl phthalocyaninato zinc(II) (oZnPcBocTyr) Boc-protected tyrosine amino acid (L-Boc-Tyr(tBu)-OH) 137 mg (0.490 mmol), 160 mg (0.490 mmol) DMTMM and (0.490 mmol) 22 lL NMM were dissolved in 5 mL dry DMF and stirred for 1 h

2.2.2. 2,3,9,10,16,17,23,24-Octa[(4-tyrosylamido)phenyl] phthalocyaninato zinc(II) (ZnPcTyr8) To a solution of 56 mg (0.016 mmol) ZnPcTyrBoc in 3 mL dry tetrahyrdofuran (THF), 10 mL trifluoracetic acid (TFA) was slowly added and stirred for 24 h at room temperature. The progress of the reaction was controlled by thin layer chromatography (TLC). The reaction mixture was poured into anhydrous diethyl ether to precipitate and filtered. The crude product was dissolved into the dilute aqueous hydrochloric acid (HCl) and then the product was re-precipitated by increasing of the pH to 9 by the addition of 5% sodium hydroxide (NaOH) solution. This method was repeated three times to obtain pure compound and the obtained product was dried under vacuum. Yield: 68%. 1H NMR (d6-DMSO), (d: ppm): 9.47 (s, 8H, NH), 8.97 (s, 8H, OH), 8.45 (s, 20H, 16  NH; 4  CH arom), 7.70 (s, 16H, CH arom), 7.23–7.15 (m, 32H, CH arom), 6.74–6.72 (m, 20H, CH arom), 4.28 (s, 8H, 4  CH, CH aliphatic), 3.16 (s, 8H, 4  CH2, CH aliphatic), 3.04 (s, 8H, 4  CH2, CH aliphatic). UV–Vis (DMSO): kmax(log e, dm3 mol 1 cm 1): 364 (2.94), 613 (2.64), 680 (3.36). IR [(KBr pellet) mmax (cm 1)]: 3392, 3201 (NH), 3014 (Aromatic CH), 2924, 2855 (Aliphatic CH), 1694, 1613 (NH), 1555, 1504, 1363, 1269, 1204, 1083, 1027, 888. Anal. Calc. for C152H128N24O24Zn: C, 66.62; H, 4.71; N, 12.27. Found: C, 66.62; H, 4.70; N, 12.26. MALDI-TOF (m/z) Calcd.: 2740.20; Found 2740.45 [M]+.

2.3. Photo-physicochemical studies 2.3.1. UV–Vis and fluorescence properties UV–Vis spectra were recorded in spectral range 300–850 nm with 2-nm resolution and 200-nm/min scan rate at room temperature using 10 mm quartz cuvette. The solutions of phthalocyanine in DMSO with variable concentrations (from 3.5 to 17.1 mM) were measured. The new ZnPcTyr8 is water-soluble. The spectra were recorded in H2O and Tris Buffer at pH = 7.4 and mixtures obtained by the addition of 2% Chremophor EL. The emission spectra were presented in spectral range between 650 and 850 nm for three excitation wavelengths (400 nm, 615 nm and 650 nm). Fluorescence quantum yield (UF) was determined in DMSO using a previously described comparative method [21]. Absorbance of the novel ZnPcTyr8 and unsubstituted Zn(II) phthalocyanine (ZnPc) used as a standard were kept under 0.05 at excitation wavelengths. The experimental study of fluorescence lifetime was carried out with time-correlated single-photon counting method (TCSPC) [22].

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

2.3.2. Singlet oxygen and photostability A photochemical method was applied to study the produced singlet oxygen in the presence of the new ZnPcTyr8 as a photosensitizer. This includes the measurement of the velocity of photooxidation of a singlet oxygen scavenger (1,3-diphenylisobenzofuran, DPBF) under red light irradiation. The sample was prepared in a cuvette and contains 2 mL DMSO with dissolved DPBF (80 mM) and ZnPcTyr8 (2 mM) under air and stirred. The standard compound ZnPc withUStd D = 0.67 of DMSO was used [20]. The changes in DPBF absorption at 415 nm during irradiation of PS with light at LED 665 nmwas recorded within time interval in which decreasing the absorbance of DPBF keep linear character. The calculations were performed by comparative method following the known formulae [23]. Photostability of the novel ZnPcTyr8 in DMSO was studied during time interval of 10 min by measurement optical density at 680 nm during irradiation with a LED 665 nm (100 mW cm 2). The homogeneity of investigated compound in solution during measurements was kept out by help of magnetic stirrer. Degree of PS photostability was determined with the rate of the photobleaching of photosensitizer. It was characterized as a rate of decreasing of molar concentration (mol s 1) in solution by irradiation following the optical density at absorption maximum. 2.4. Photocytotoxicity study 2.4.1. Cell lines and culture conditions Two cancer cell lines which are recognized as highly invasive a basal type adenocarcinoma (MDA-MB-231) and a low invasive luminal-like ductal adenocarcinoma (MCF-7) were studied in comparison to a model healthy breast tissue such as the epithelial cell line MCF-10A. The experimental cells were cultured (Dulbecco’s Modified Eagle’s Medium – high glucose, DMEM, Sigma), and supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml) from Lonza. The growth medium for MCF-7 cell line was of 0.01 mg/mL human recombinant insulin and 1% Non Essential Amino Acids (NEAA) by addition of the above. In case of MCF-10A was added 0.01 mg/mL human recombinant insulin, 100 ng/mL cholera toxin, 10 ng/mL human Epidermal Growth Factor (hEGF) and 500 ng/mL hydrocortisone. The model cells were grown in 25 cm2 tissue culture flasks (Greiner Bio-One) and maintained in a moisten atmosphere (5% CO2 at 37 °C). 2.4.2. Light source The light source used for in vitro experiments was a lamp with wavelength at 665 nm consisting of a template with 25 LED bulbs arranged in a circle. The diameter of 5 cm was possible to irradiate. This LED device can reach a light output intensity up to 60 mW cm 2 on area of 50 cm 2 and with a spectral width of 25 nm. The power density distribution was arranged according to the distance of the light source from the cell plates in order to provide a homogeneous distribution of the required power density. A dose of 50 J cm 2 is obtained for power density of 60 mW cm 2 and almost 15 min irradiation time. 2.4.3. In vitro PDT study The cells with a density of 5  103 cells in 100 mL were placed in each well of 96-well flat-bottomed plates to allow the adherence for 24 h before incubation with ZnPcs. The culture medium was replaced after formation of the stable mono-layer of cells. In each well the studied Zn(II) phthalocyanines was added with two-fold decreasing concentration starting from 20 mM to 0.15 mM. The incubation (3 h) was carried out under the same conditions, and then the culture medium was again aspirated and replaced. The plates were then illuminated for 30 min using LED matrix light

123

source. The power density distribution was measured at different distances of the light source from the plates in order to provide a homogeneous distribution of power density on irradiated area. The evaluations of PDT activities were performed in comparison by measuring the cell viability using Neutral Red Uptake assay (NRU assay). After 72 h of incubation in a thermostat (5% CO2 at 37 °C), the obtained data were analyzed. The photodynamic effect is expressed as a percentage of the untreated control groups of cells calculated for each concentration. The statistical analysis included one-way ANOVA analysis of variance followed by Bonferroni’s post-hoc test. The average results of the four wells for each sample were calculated. Each experiment was carried out in triplicate and the results were presented as mean ± standard deviation (SD). 3. Results and discussion 3.1. Synthesis A novel zinc(II) phthalocyanine bearing eight L-tyrosyl moieties was newly synthesized according to the synthetic pathway presented in Scheme 1. The synthetic procedure involves five steps which starts with synthesis of 4,5-bis(4-nitrophenoxy) phthalonitrile according to previously described reaction procedure [19,20]. Next step of procedure undergoes cyclotetramerization of the obtained 4,5-bis(4-nitrophenoxy) phthalonitrile in the presence of anhydrous zinc(II) acetate and a catalyst in 1-pentanol by refluxing to afford zinc(II) phthalocyanine bearing eight p-nitrophenoxy substituents (Scheme 1). The reduction of eight nitro groups of oZnPcNO2 to amino groups was carried out by reaction with sodium sulfide nonahydrate in dry DMF at 65 °C [24–26]. This resulted to octa-aminophenoxy substituted phthalocyanine (oZnPcNH2) which was used for reaction of conjugation with protected tyrosine (L-BocTyr(tBu)-OH). The reaction of L-BocTyr(tBu)OH with oZnPcNH2 was carried out in the presence of the coupling reagents (DMTMM and NMM). The phthalocyanine oZnPcBocTyr (tBu) was obtained with high purity and yield. The protected compound oZnPcBocTyr(tBu) was dissolved in THF and TFA was added. Both protection groups namely tert-butoxycarbonyl- (BOC) and O-tert-butyl- (-tBu) were removed by stirring at room temperature. The final phthalocyanine substituted with eight tyrosyl groups (ZnPcTyr8) was evaluated as a water-soluble phthalocyanine. The good solubility was observed in several organic solvents (methanol, ethanol, DMF and DMSO) but lack of solubility of ZnPcTyr8 in DCM, Chloroform, acetone and THF. ZnPcTyr8 was analyzed by the known spectroscopic techniques 1H NMR, FT-IR, MALDI-TOF, UV–Vis and elemental analysis. 1H NMR analysis in DMSO-d6 showed significant aggregation for the both newly synthesized phthalocyanines. The presence of additional aromatic rings, compared to other tyrosine containing structures [27], results in spectra with broad signals, even at high temperature, thus hampering the precise assignment of the spectra. 3.2. Photo-physicochemical properties The absorbance of the new water-soluble ZnPcTyr8 was studied in DMSO solutions with different concentrations (Fig. 1a). The absorption spectra were recorded with the typical sharp Q band with maximum at 680 nm in DMSO. This suggests that for concentrations up to 17.1 lM ZnPcTyr8 exists as monomeric molecules. The spectra show an additional absorption B band at 356 nm with a half of the intensity of the Q band. The inserted linear calibration plot shows the dependence of the absorption intensity in the studied concentration range (Fig. 1a). The observed dependence is linear and followed the Buge-Lambert-Beer low. The absorption

124

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

Scheme 1. Synthetic route for preparation 2,3,9,10,16,17,23,24-octa[(4-tyrosylamido) phenyl] phthalocyanine zinc(II) (ZnPcTyr8).

spectra of water-soluble ZnPcTyr8 also were recorded in water and in buffer solution at pH 7.4 and for the both media were observed low intensity broad maxima at 636 nm and 682 nm (Fig. 1b) due to the formation of physical aggregates which is typical for phthalocyanines in aqua media and disaggregation process occurs by addition of 2% Chremophor EL (Fig. 1b). As can be seen the position of the absorption maxima are the same independently on the solvent’s polarity. The values of photophysical parameters are summarized in Table 1. The absorption spectra showed typical band around 635 nm. The lower absorbance of the Q band was observed in ZnPcTyr8 in water which suggests aggregation in aqua media. In contrast of tetra-tyrosine substituted ZnPcTyr, newly synthesized ZnPcTyr8 is water-soluble phthalocyanine and in polar media tend easily to form aggregates (Fig. 1b). Previously reported tetra-tyrosyl substituted ZnPcTyr was studied with the characteristic spectra for monomeric phthalocyanines in DMF and the formation of aggregates occur in polar solvents such as EtOH, MeOH and water [16,18]. Fluorescence emission spectra of ZnPcTyr8 were recorded with different excitation wavelengths at 400, 615 and 650 nm in DMSO, water and Tris Buffer at pH = 7.4 (Fig. 2). They showed emission maxima at 693 nm in DMSO and 688 nm in water and respectively

which are bathochromicaly shifted with 13 nm in DMSO and 8 nm in water and buffer compared to the Q bands in the absorption spectra of (Fig. 2a). Fluorescence emission spectra of ZnPcTyr8 recorded at 400 nm and 650 nm in DMSO showed identical position of fluorescence maximum suggesting the monomeric state of ZnPcTyr8 as well as the lack of any changes in symmetry of molecules. The spectra of absorption and fluorescence excitation and emission of ZnPcTyr8 in dH2O by addition of 2% Cremophor EL are compared in Fig. 2b. The fluorescence quantum yield (UF) of the ZnPcTyr8 in DMSO is relatively low (UF = 0.04) suggesting more the quenching of the singlet state by the triplet state and non-radiated transitions. The evaluation of the fluorescence lifetime (sF) is of importance to PDT process because this is the time the molecules keep are in their excited state and being before returning to the ground state. The sF – value of 2.05 ns (CHISQ: 1.810) for ZnPcTyr8 in DMSO was determined by a time correlated single photon counting (TCSPC) method. As can be seen the decay fluorescence curve has a monoexponential decreasing (Fig. 3). This is suggests that only one fluorescence molecule exists in the solution. The fluorescence lifetime of the recently studied tetra-substituted ZnPcTyr with 3.01 ns (CHISQ: 1.011) (Table 1) was evaluated with lower value than

125

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

Fig. 2. Fluorescence spectra of ZnPcTyr8 in DMSO at kex = 400 and 650 nm in water and Tris Buffer at pH = 7.4 at kex: 615 nm (a) and absorption, excitation and emission spectra of ZnPcTyr8 in dH2O + 2% Cremophor EL (b). Fig. 1. Absorption spectra of ZnPcTyr8 in DMSO for concentration range of 3.5– 17.1 mM (a); Normalized absorption spectra of ZnPcTyr8 in DMSO, dH2O, dH2O + 2% Cremophor EL, Tris Buffer pH = 7.4, Tris Buffer pH = 7.4 + 2% Cremophor EL (b).

the unsubstituted ZnPc (3.64 ns). This observation confirms that the addition of any substituents can physically quench the shortliving singlet oxygen. The capacity of singlet oxygen production by the new tyrosine conjugated ZnPc was evaluated using a chemical scavenger (DPBF) of the generated singlet oxygen as a result of irradiation of the studied ZnPcTyr8 as well as ZnPcTyr in DMSO (Fig. 4). The photosensitization reaction mechanism type II which involves generation of molecular singlet oxygen, occurs with participation of the phthalocyanines [24]. The value of quantum yield of the highly reactive singlet oxygen is very important parameter for evaluation of the efficiency of tyrosine bounded to photosensitizer in photodynamic process [25]. The newly synthesized ZnPcTyr8 showed sufficient level of singlet oxygen quantum yield (0.38) compared to tetra tyrosine- containing ZnPcTyr (0.63) during local light exposure. Moreover the formation of molecular singlet oxygen does not affect the Q band intensity of ZnPcTyr8 which suggests the lack of photooxidative effect on molecule by irradiation. The singlet oxygen quantum yield of ZnPcTyr8 (UD = 0.38) can be evaluated as characteristic for metal phthalocyanine complexes [27].

Fig. 3. Time-resolved fluorescence curve of ZnPcTyr8 in DMSO at kex: 674 nm laser.

The option of singlet oxygen physical quenching is more probable by the eight peripheral substituents than four aminophenoxy-tyrosyl groups on ZnPc ring molecule. The physical quenching by bulky

Table 1 Photophysical properties of ZnPcTyr and ZnPcTyr8 in DMSO. PS

Q-band kmax, (nm)

(log e)

Soret band kmax (nm)

(log e)

Excitation kex, (nm)

Emission kEm, (nm)

Stokes shift DStokes, (nm)

UF

sF (ns)

ZnPcTyr8 ZnPcTyr

680 nm 613 nm 682 nm

4.36 3.64 4.94

364 357

3.94 2.31

684 nm 682 nm

693 nm 690 nm

13 nm 8 nm

0.041 0.1

2.05 ns 3.01 ns

126

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

substituents was known to diminish the singlet oxygen quantum yields of some other substituted phthalocyanines [28]. The photostability is an essential property for photosensitizer during the illumination period. The photobleaching rates of the tyrosine conjugated ZnPcs were evaluated by irradiation in DMSO solutions. The photostability was calculated with photobleaching rates of 32  10 10 M s 1 for ZnPcTyr8 and 3.1  10 10 M s 1 for ZnPcTyr (Fig. 5). The obtained values are higher compared to standard compound ZnPc with rate of 0.94  10 10 M s 1. Increasing the photobleaching rate from tetra- and octa tyrosine ZnPcs versus ZnPc may be effected from the number of the substituted tyrosine groups which tend to lower the photostability. The study of the rate of photobleaching of the newly synthesized ZnPcTyr8 suggests optimal photostability without formation of by-products due to power density of the applied light. The collapse of the absorption spectra without any distortion of the shape confirms that photobleaching is not associated with phototransformation of phthalocyanine molecules in the visible region.

cancer cells (MDA-MB-231 and MCF-7) and one normal cell line (MCF-10A). The Both phthalocyanines (ZnPcTyr8 and ZnPcTyr) were studied for wide concentration range (0.15–20 mM) after irradiation with specific LED 665 nm and light dose of 50 J cm 2 (Fig. 6a–c). The study showed the lack of dark toxicity of ZnPcTyr8 as well as for ZnPcTyr for tested tumor and healthy cells. In comparison the unsubstituted ZnPc was also studied following the same treatment conditions. The Both tyrosine- conjugated ZnPcs

3.3. In vitro PDT activity Photocytotoxicity assays were carried out with two tyrosineconjugated ZnPcs versus unsubstituted ZnPc on two human breast

Fig. 4. Exponential curves at absorption maximum (417 nm) of singlet oxygen scavenger 1,3-diphenylisobenzofuran (DPBF) measured in the presence of 1.5 mM ZnPcTyr8, ZnPcTyr and ZnPc in DMSO during irradiation with LED 637 nm.

Fig. 5. Photobleaching kinetic curves for 1.8 mM ZnPcTyr8, ZnPcTyr and ZnPc in DMSO during irradiation with LED 637 nm. Inset: values of the photobleaching rate.

Fig. 6. Viability of tumor cell lines MCF-7 (a) and MDA-MB-231 (b) and normal cell line MCF-10A (c) for 0.15–20.00 mM ZnPcTyr8, ZnPcTyr and ZnPc at light dose of 50 J cm 2.

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

showed a gentle phototoxicity with efficacy at high concentrations (>20 lM). The strong phototoxic effects for ZnPc was observed at low concentrations (0.15–10 lM). However harsh photocytotoxic effect of the basic was observed for ZnPc was but also for the normal cell line (MCF-10). The tendency in the photocytotoxicity was similar for the both tyrosine substituted ZnPcs namely low photoinactivation for concentrations up to 10 lM with trend of moderate photocytotoxic effect at higher concentrations (>10 lM). The studied tumor cell lines were evaluated with 37% viability for MCF-7 cells and 50% for MDA-MB-231 cells for the studied tyrosine conjugated ZnPcs. This observation of higher efficiency against MDA-MB-231 cells can be explained by the aggressive nature of the line of MCF-7 cells. As can be seen the healthy cells (MCF-10A) are non-affected for concentrations between 1–10 lM ZnPcTyr8 and ZnPcTyr but for the same conditions the unsubstituted ZnPc shows strong photocytotoxic effect (Fig. 6c). A negligible inactivation of normal cells of MCF-10A (80% viability) was determined at concentrations >10 lM for both ZnPcs with four and eight tyrosine moieties. The recent knowledge about porphyrin with amino acid suggests that these conjugates are PDT inactive until they reached the cancerous cells [29]. The basic molecule seems to be active in the cancer cells due to cleavage of the peptide sequence by MMP enzymes in cancer cells. These MMP enzymes are secreted at a limited level in normal cells. According to recent results, intermolecular energy transfer occurred in synthesized novel conjugates and they could be passive until they reach the target cancer tissue [30]. The synthesized novel phthalocyanine bioconjugates may serve as a promising approach in development of new generation photo-activatable photosensitizers with gentle photocytotoxic action to malignances, minimal or lack of phototoxicity to the normal surrounding tissue, and finally with selective photodynamic therapy response.

4. Conclusion Novel octa-(4-tyrosylamido) phenyl zinc(II) phthalocyanine (ZnPcTyr8) was synthesized in good yield and purity as observed by chemical analyses (1H NMR, FT-IR, UV–Vis, elemental analysis and mass). The connection of eight tyrosine moieties to hydrophobic macrocycle of Zn(II) phthalocyanine leads to water solubility of new ZnPcTyr8. The absorption spectrum of ZnPcTyr8 shows Qband at 680 nm in DMSO which is red shifted compared to unsubstituted ZnPc (671 nm). The fluorescence spectra of ZnPcTyr8 in DMSO shows small bathochromic shift to the red region (13 nm) with low fluorescence quantum yield (0.04) which is lower than ZnPcTyr (0.1). Singlet oxygen quantum yields of the new ZnPcTyr8 (0.38) is also lower than the yield of ZnPcTyr (0.63) in DMSO. The Both conjugated ZnPcs with eight and four tyrosine units show faster photobleaching rates than the value of unsubstituted ZnPc during irradiation time of PDT routine. Additionally, in vitro studies with ZnPcTyr8 and ZnPcTyr showed gentle phototoxic effect on tumor as well as on normal cells while the strong photocytotoxic effect was observed for unsubstituted ZnPc. The new tyrosine- conjugated ZnPcTyr8 characterizes with water solubility, proper photophysicochemical properties but moderate photoinactivation efficacy which can be a promising feature for superficial PDT applications.

Acknowledgments MA thanks to the project for career development of young scientists (BAS, DFNP-150). The support made by of the National Science Fund (B02/9/2014, Bulgaria).

127

Declaration of interest The authors declare that they have no conflict of interest. References [1] X. Li, S. Lee, J. Yoon, Supramolecular photosensitizers rejuvenate photodynamic therapy, Chem. Soc. Rev. 47 (2018) 1174. [2] D.E. Dolmans, D. Fukumura, R.K. Jain, Photodynamic therapy for cancer, Nat. Rev. Cancer 3 (2003) 380. [3] S. Hackbarth, J. Schlothauer, A. Preub, B. Röder, New insights to primary photodynamic effects – singlet oxygen kinetics in living cells, J. Photochem. Photobiol. B: Biol. 98 (2010) 173. [4] F. Moret, E. Reddi, Strategies for optimizing the delivery to tumors of macrocyclic photosensitizers used in photodynamic therapy (PDT), J. Porphy. Phthal. 21 (2017) 1. [5] T. Goslinski, T. Osmalek, K. Konopka, M. Wierzchowski, P. Fita, J. Mielcarek, Photophysical properties and photocytotoxicity of novel phthalocyanines – potentially useful for their application in photodynamic therapy, Polyhedron 30 (2011) 1538. [6] A.M. De Pinilos Bayona, P. Mroz, C. Thunshelle, M.R. Hamblin, Design features for optimization of tetrapyrroles macrocycles as antimicrobial and anticancer photosensitizers, Chem. Biol. Drugs Des. 89 (2017) 192. [7] Z. Chen, S. Zhou, J. Chen, Y. Deng, Z. Luo, H. Chen, M.R. Hamblin, M. Huang, Pentalysine beta-carbonylphthalocyanine zink: an effective tumor-targeting photosensitizer for photodynamic therapy, ChemMedChem 5 (6) (2010) 890. [8] , Handbook of Porphyrin Science – The Key Role of Peripheral Substituents in Chemistry of Phthalocyanines vol. 3 (2010) 2. [9] F. Li, Q. Liu, Z. Liang, J. Wang, M. Pang, W. Huang, W. Wu, Z. Hong, Synthesis and biological evaluation of peptide-conjugated phthalocyanine photosensitizers with hydrophilic modifications, Org. Biomol. Chem. 13 (2016) 3409. [10] M.R. Ke, J.M. Eastel, K.L.K. Ngai, Y.Y. Cheung, P.K.S. Chan, M. Hui, D.K.P. Ng, P.C. Lo, Oligolysine-conjugated zinc(II) phthalocyanines as efficient photosensitizers for antimicrobial photodynamic therapy, Chem. Asian J. 9 (2014) 1868. [11] R. Wang, L. Zhou, K. Zhou, J. Sun, Sh. Jiang, Wei, Positively charged phthalocyanine-arginine conjugates as efficient photosensitizer for photodynamic therapy, Bioorg. Med. Chem. 25 (5) (2017) 1643. [12] H.Y. Cheah, E. Gallon, F. Dumoulin, S.Z. Hoe, N. Japundzic-Zigon, S. Glumac, H.B. Lee, P. Anand, L.Y. Chung, M.J. Vicent, L.V. Kiew, Near-infrared activatable phthalocyanine-poly-L-glutamic acid conjugate: enhanced in vivo safety and antitumor efficacy toward an effective photodynamic cancer therapy, Mol. Pharm. 15 (7) (2018) 2594. [13] M. Goksel, M. Durmus, D. Atilla, A comparative study on photophysical and photochemical properties of zinc phthalocyanines with different molecular symmetries, J. Porphy. Phthal. 16 (7–8) (2012) 895. [14] E. Ranyuk, N. Cauchon, K. Klarksov, B. Guerin, J.E. van Lier, Phthalocyaninepeptide conjugates: receptor-targeting bifunctional agents for imaging and photodynamic therapy, J. Med. Chem. 56 (2013) 1520. [15] O. Glushkovskaya, O.J. Kurths, E. Borisova, S. Sokolovski, V. Mantareva, I. Angelov, et al., Photodynamic opening of blood-brain barrier, Biomed. Opt. Express 8 (11) (2017) 5040. [16] M. Aliosman, M. Goksel, V. Mantareva, I. Stoineva, M. Durmus, Tyrosine conjugated zinc(II) phthalocyanine for photodynamic therapy: synthesis and photophysicochemical properties, J. Photochem. Photobiol. A: Chem. 133 (2017) 101. [17] M. Aliosman, I. Eneva, I. Stoineva, M. Durmus, V. Mantareva, Aminophenoxysubstituted zinc(II) phthalocyanines as basic photosensitizers for conjugation with biologically active moieties via amide bond, Bulg. Chem. Comm. (Special issue E) (2017) 79. [18] V. Mantareva, M. Aliosman, M. Durmus, I. Angelov, Amino acids substituted phthalocyanine complexes: an overview on the synthesis and UV-vis spectral properties related to photodynamic strategy, Bulg. Chem. Comm. 50 (Special issue – D) (2018) 185. [19] D. Wöhrle, M. Eskes, K. Shigehara, A. Yamada, A simple synthesis of 4,5disubstituted 1,2-dicyanobenzenes and 2,3,9,10,16,17,23,24-octasubstituted phthalocyanines, Synthesis 2 (1993) 194. [20] S.E. Maree, T. Nyokong, Syntheses and photochemical properties of octasubstituted phthalocyaninato zinc complexes, J. Porphy. Phthal. 5 (2001) 782. [21] G.C. Taskin, M. Durmusß, F. Yuksel, V. Mantareva, V. Kussovski, I. Angelov, D. Atilla, Axially parabens substituted silicon (IV) phthalocyanines towards dental pathogen Streptococcus mutans: synthesis, photophysical, photochemical and in vitro properties, J. Photochem. Photobiol. A: Chem. 306 (2015) 31. [22] B.Y. Mikhail, S. Achilefu, Fluorescence Lifetime Measurements and Biological Imaging, Chem. Rev. 110 (2010) 2641. [23] A. Ogunsipe, D. Maree, T. Nyokong, Solvent effects on the photochemical and fluorescence properties of zinc phthalocyanine derivatives, J. Mol. Struct. 650 (2003) 131. [24] S.V. Kudrevıch, H. Ali, J.E. van Lier, Syntheses of monosulfonated phthalocyanines, benzonaphthoporphyrazines and porphyrins via the Meerwein reaction, J. Chem. Soc. Perkin Trans. 1 (1994) 2767.

128

M. Aliosman et al. / Polyhedron 162 (2019) 121–128

[25] F. Cong, B. Ning, X. Du, C. Ma, H. Yu, B. Chen, Facile synthesis, characterization and property comparisons of tetraamino-metallophthalocyanines with and without intramolecular hydrogen bonds, Dyes Pigm. 66 (2005) 149. [26] L. Wang, L. Gui, S. Lu, L. Zhou, J. Zhou, S. Wei, Tumor microenvironmentresponsive charge reversal zinc phthalocyanines based on amino acids for photodynamic therapy, Dyes Pigm. 126 (2016) 239. [27] B. Kiyak, A.A. Esenpinar, M. Balut, Syntesis, characterization, photophysical and photochemical properties of zinc and indium phthalocyanines bearing a vanillylacetone moiety known as an anticancer agent, Polyhedron 90 (18) (2015) 183.

[28] N.Sh. Lebedeva, Y.A. Gubarev, A.I. Vyugin, O.I. Koifman, Investigation of interaction between alkoxy substituted phthalocyanines with different lengths of alkyl residue and bovine serum albumin, J. Luminescence 166 (2015) 71. [29] H.M. Wang, J.Q. Jiang, J.H. Xiao, R.L. Gao, F.Y. Lin, X.Y. Liu, Porphyrin with amino acid moieties: a tumor photosensitizer, Chem.-Biol. Interact. 172 (2008) 154. [30] P. Singh, A. Kumar, S. Kaur, A. Singh, M. Gupta, G. Kaur, Stitching of tyrosine and 10H-acridin-9-one: Turn-ON fluorescence in the narrow pH range 7.4–8.5 and intracellular labelling of cancer cells, Med. Chem. Commun. 7 (2016) 632.