In vitro evaluation of Schiff base-decorated phthalocyanines for photodynamic therapy in PC3 prostate cancer cells

Abstract

Schiff base-decorated non-ionic phthalocyanine compounds with different metals and a metal-free derivative were synthesized and characterized using different spectroscopic techniques. The influence of the central metal on the photophysicochemical properties was systematically investigated in DMSO. The obtained results reveal that the singlet oxygen quantum yield followed the order In3b > Zn3b > 3bH₂Pc in DMSO. The synthesized non-ionic phthalocyanine compounds, except metal-free phthalocyanine (0.12 for 3bH₂Pc), have good Φ_Δ values (0.71 for Zn3b and 0.85 for In3b) compared with the unsubstituted ZnPc in DMSO (Φ_Δ = 0.67). In addition to the photophysicochemical studies, in vitro PDT studies were also performed on PC3 prostate cancer cells to evaluate the biological activity of Pcs. All compounds exhibited low dark cytotoxicity at the selected concentration. Upon light irradiation, phthalocyanine-mediated PDT significantly reduced cell viability and induced apoptotic cell death. These effects were accompanied by a pronounced increase in intracellular ROS generation, particularly in the cells treated with metallophthalocyanine-mediated PDT. In particular, In3b- and Zn3b-mediated PDT exhibited markedly enhanced cytotoxic and apoptotic effects compared with metal-free 3bH₂Pc. These findings demonstrate a strong correlation between improved photophysical properties, increased ROS generation, and enhanced in vitro PDT efficacy.

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1. Introduction

Cancer, a disease with multistep processes, is one of the most life-threatening diseases affecting humans. According to GLOBOCAN data, lung cancer was estimated to have caused the greatest number of cancer-related deaths in 2023.¹ Photodynamic therapy (PDT), which is a non-invasive and laser-controlled cancer treatment method, has been successfully applied to different cancer cell types, such as prostate cancer,² breast cancer,³ colon cancer,⁴ lung cancer⁵ and melanoma.⁶ The three main components necessary for PDT application are molecular oxygen, photosensitizer and light. In PDT, malignant tumors are destroyed with reactive oxygen species (ROS), which are obtained as a result of photochemical reactions.^7,8 Lipophilic phthalocyanines (Pcs), that are planar and aromatic and are derived by the linking of different substituents and metals, provide various distinct properties. Due to these properties, suitable phthalocyanines have been synthesized and used for numerous applications like catalysis, gas sensors, photodynamic therapy, electrochromism, liquid crystal, and solar cells.^9–15 The presence of diamagnetic metals, such as Zn(II), Al(III), Ga(III), Mg(II) and In(III), in the central cavity of phthalocyanine compounds increases the generation of singlet oxygen and the triplet lifetime.^16–20 Schiff bases, also called imines, which play an essential role in coordination chemistry, are synthesized by the formation of an azomethine bond (–N=CH–) as a result of the condensation reaction between carbonyl compounds and primary amines. These types of compounds are extensively employed in pharmaceutical and biological sciences owing to their diverse chemical and biological properties. Among the biological properties, their pronounced antitumoral activity makes them attractive candidates for anticancer applications, particularly in photodynamic therapy (PDT).^21–28

While phthalocyanines are well-known photosensitizers with strong absorption in the therapeutic window and efficient singlet oxygen generation, Schiff base moieties contribute additional electronic modulation and intrinsic biological activity. Furthermore, the incorporation of diamagnetic metal ions, such as Zn(II) and In(III), can promote intersystem crossing and improve triplet state formation, thereby enhancing the generation of reactive oxygen species. Therefore, the design of imine-functionalized metal-free and metallophthalocyanines provides a promising platform for the development of advanced PDT agents.

In this study, novel metal-free and metallophthalocyanines (Zn(II) and In(III)) bearing imine segments were successfully synthesized and comprehensively characterized for the first time. Their photophysicochemical properties, including fluorescence, singlet oxygen generation, and photodegradation quantum yields, were systematically investigated in DMSO. By directly correlating these photophysicochemical parameters with in vitro PDT results, the present work provides a comprehensive evaluation of imine-substituted metal-free and metallophthalocyanines. The comparative analysis clearly demonstrates the critical role of the central metal ion in determining the PDT efficiency, offering valuable insights for the rational design of imine-based photosensitizers with improved therapeutic performance.

2. Experimental

The synthetic pathway for the new peripherally Schiff base-decorated phthalocyanine complexes (3bH₂Pc, Zn3b and In3b) is shown in Scheme 1. The Schiff base derivative 3a was synthesized via a condensation reaction between 4-methoxybenzaldehyde and 4-aminophenol using acetic acid as a catalyst. After that, 3a reacted with 4-nitrophthalonitrile under basic conditions to yield the phthalonitrile derivative 3b. Schiff base-decorated complexes (3bH₂Pc, Zn3b and In3b) were obtained by the tetramerization of the phthalonitrile derivative 3b with related metal salts in the presence of a DBU catalyst.

Scheme 1 Synthetic pathway of the imine-substituted phthalocyanines (3bH₂Pc, Zn3b, and In3b): (i) dry ethanol and glacial acetic acid at 78 °C; (ii) K₂CO₃ and dry DMF at 60 °C and (iii) no metal salt for 3bH₂Pc, Zn(Ac)₂ for Zn3b, and InCl₃ for In3b, n-pentanol, and DBU at 145 °C.

2.1. Synthesis of 4-[(E)-{[4-methoxyphenyl]methylidene}amino]phenol (3a)

Compound 3a was synthesized by using the method reported in the literature.²⁹ A solution of 2 mL of 4-methoxybenzaldehyde was added to a solution of 4-aminophenol (1.045 g, 9.5 mmol) in 12.5 mL dry ethanol in a two-necked round-bottomed flask. After adding 5 drops of glacial acetic acid, the mixture was refluxed at 78 °C for 3.5 hours under an argon atmosphere. The obtained white product was filtered and washed with cold methanol, followed by drying the precipitate in a vacuum over P₂O₅. Yield: 1.56 g (71%). M.P.: 188 °C–189 °C. Anal. calc. for C₁₄H₁₃NO₂ (%): C, 73.99; H, 5.77; N, 6.16. Found (%): C, 73.96, H, 5.79, N, 6.12. FT-IR ν_max/cm⁻¹: 3058 (Ar–CH), 2979–2847 (Aliph. –CH), 1604 (C=N), 1510, 1447 (C=C). ¹H-NMR (CDCl₃), (δ: ppm) = 9.82 (s, 1 Ar–OH), 8.57 (s, 1 HC=N), 7.78 (d, J ≈ 8.7 Hz; 2 Ar–H), 7.40 (d, J ≈ 7.0 Hz; 2 Ar–H), 7.07 (d, J ≈ 7.7 Hz; 2 Ar–H), 6.75 (d, J ≈ 8.8 Hz; 2 Ar–H), 3.83 (s, 3H, O–CH₃). MS (MALDI-MS) m/z: calc. 227.26; found: 227.088 [M]⁺.

2.2. Synthesis of 4-{4-[(E)-{[4-methoxyphenyl]methylidene}amino]phenoxy}benzene-1,2-dicarbonitrile (3b)

The Schiff base ligand (3a) (1.29 g, 5.7 mmol) and 12.5 mL dry DMF were dissolved in a two-necked round-bottomed flask. After adding the solution of 4-nitrophthalonitrile (0.98 g, 5.7 mmol) to 6 mL dry DMF, the mixture was stirred for 20 min at 60 °C. Anhydrous potassium carbonate (1.61 g, 11.6 mmol) was added portion-wise to the reaction mixture for two hours under an argon atmosphere. The reaction mixture was stirred for 24 hours at 60 °C under an argon atmosphere. At the end of the reaction, the mixture was poured into a 500 mL ice-water (50 : 50) solution and collected by centrifugation. The collected precipitate was crystallized in acetone and washed with cold acetone. Then, the pure product was dried under vacuum over P₂O₅. Yield: 1.25 g (62%). M.P.: 152 °C–153 °C. Anal. calc. for C₂₂H₁₅N₃O₂ (%): C, 74.78; H, 4.28; N, 11.89. Found (%): C, 74.80, H, 4.25, N, 11.84. FT-IR ν_max/cm⁻¹: 3077 (Ar–H), 2971–2839 (Aliph. C–H), 2230 (C≡N), 1600 (C=N), 1567, 1508, 1481 (C=C), 1284, 1090 (C–O–C). ¹H-NMR (500 MHz, CDCl₃): δ (ppm) = 8.44 (s, 1 H–C=N), 7.90 (d, J ≈ 7.6 Hz; 2 Ar–H), 7.75 (d, J ≈ 8.7 Hz; 1 Ar–H), 7.32–7.27 (m, 4 Ar–H), 7.12 (d, J ≈ 8.5 Hz; 2 Ar–H), 7.03 (d, J ≈ 8.5 Hz; 2 Ar–H), 3.92 (s, 3H, CH₃). ¹³C-NMR (126 MHz, CDCl₃): δ (ppm) = 162.57, 162.06, 160.33, 151.14, 150.56, 135.43, 130.69, 128.94, 122.97, 121.38, 121.37, 121.28, 117.69, 115.42, 114.99, 114.32, 108.76, 55.50. MS (MALDI-MS) m/z: calc. 353.37; found: 353.02 [M]⁺.

2.3. General synthesis method of the phthalocyanine compounds (3bH₂Pc, Zn3b, and In3b)

A mixture of compound 3b (0.11 g, 0.31 mmol for 3bH₂Pc and 0.10 g, 0.29 mmol for Zn3b and In3b), metal salt (0.017 g, 0.07 mmol anhydrous zinc(II) acetate and indium(III) chloride for Zn3b and In3b, respectively) and 3 drops of DBU in n-pentanol (3 mL) was stirred at 145 °C for 5 h under argon atmosphere in a Schlenk tube. Metal salt was not used in the synthesis of metal-free phthalocyanine (3bH₂Pc). When the reaction mixture was cooled to room temperature, it was precipitated using 200 mL n-hexane and then centrifuged. Subsequently, the crude product obtained was purified by column chromatography on silica gel using THF as the eluent.

2.3.1. 2(3),9(10),16(17),23(24)-Tetrakis-4-{4-[(E)-{[4-methoxyphenyl]methylidene}amino]phenoxy} phthalocyanine (3bH₂Pc). Yield: 0.093 g (21%). Anal. calc. for C₈₈H₆₂N₁₂O₈ (%): C, 74.67; H, 4.41; N, 11.87. Found (%): C, 74.59, H, 4.38, N, 11.82. UV-vis (DMSO) λ_max/nm (log ε): 703 (3.56), 674 (3.61), 641 (3.38), 333 (4.53). FT-IR ν_max/cm⁻¹: 3288 (–NH), 3065 (Ar–H), 2930, 2858 (Aliph. C–H), 1602 (C=N), 1226, 1025 (C–O–C). ¹H-NMR (DMSO-d₆), (δ: ppm) = 8.6 (s, 4 HC=N), 8.7–6.6 (m, 44 Ar–H), 3.84 (s, 12 H, 4 CH₃). MS (MALDI-MS) m/z: calc. 1415.51; found: 1415.20 [M]⁺.

2.3.2. 2(3),9(10),16(17),23(24)-Tetrakis-4-{4-[(E)-{[4-methoxyphenyl]methylidene}amino]phenoxy} phthalocyaninato zinc(II) (Zn3b). Yield: 0.08 g (19%). Anal. calc. for C₈₈H₆₀N₁₂O₈Zn (%): C, 71.47; H, 4.09; N, 11.37. Found (%): C, 71.18, H, 4.03, N, 11.30. UV-vis (DMSO) λ_max/nm (log ε): 682 (4.80), 615 (4.14), 348 (4.66). FT-IR ν_max/cm⁻¹: 3062 (Ar–H), 2929, 2839 (Aliph. C–H), 1603 (C=N), 1226, 1089 (C–O–C); ¹H-NMR (DMSO-d₆), (δ: ppm) = 8.8–6.8 (m, 4 HC=N, 44 Ar–H), 3.84 (s, 12 H, 4 CH₃). MS (MALDI-MS) m/z: calc. 1478.87; found: 1478.944 [M]⁺.

2.3.3. 2(3),9(10),16(17),23(24)-Tetrakis-4-{4-[(E)-{[4-methoxyphenyl]methylidene}amino]phenoxy} phthalocyaninato chloroindium(III) (In3b). Yield: 0.037 g (8%). Anal. calc. for C₈₈H₆₀N₁₂O₈InCl (%): C, 67.59; H, 3.87; N, 10.75. Found (%): C, 67.32, H, 3.79, N, 10.74. UV-vis (DMSO) λ_max/nm (log ε): 696 (4.16), 624 (3.95), 340 (4.34). FT-IR ν_max/cm⁻¹: 3063 (Ar–H), 2930, 2864 (Aliph. C–H), 1602 (C=N), 1224, 1080 (C–O–C). ¹H-NMR (DMSO-d₆), (δ: ppm) = 8.58 (s, 4 HC=N), 7.9–6.6 (m, 44 Ar–H), 3.84 (s, 12 H, 4 CH₃). MS (MALDI-MS) m/z: calc. 1563.76; found: 1526.35 [M − Cl − 2H]⁺.

3. Results and discussion

3.1. Synthesis and characterization

Compound 3a was synthesized via the condensation reaction between 4-aminophenol and 4-methoxybenzaldehyde. In the presence of K₂CO₃, the starting phthalonitrile compound (3b) was synthesized via the nucleophilic aromatic substitution reaction between 3a and 4-nitrophthalonitrile in basic media (DMF). Afterwards, a metal-free phthalocyanine compound was obtained with the cyclotetramerization of phthalonitrile compound 3b in n-pentanol and DBU. Unlike the metal-free phthalocyanine, the synthesis of Zn3b and In3b was carried out in the presence of Zn(CH₃COO)₂ and InCl₃, respectively. UV-vis, FT-IR, ¹H-NMR, MALDI-TOF and elemental analysis techniques were used to characterize the structures of all the synthesized novel compounds. The synthetic routes of 3a, 3b, 3bH₂Pc, Zn3b and In3b are given in Scheme 1.

In the FT-IR spectrum of compound 3a, the azomethine (–C=N) peak was observed at 1604 cm⁻¹. The C≡N and C=N vibrations at 2230 and 1600 cm⁻¹, respectively, were observed upon the formation of the phthalonitrile compound 3b. After the cyclotetramerization of phthalonitrile, the disappearance of the specific C≡N vibration confirms the synthesis of 3bH₂Pc, Zn3b and In3b complexes. Also, the observed peak at 3288 cm⁻¹ corresponds to the specific inner core –NH band of compound 3bH₂Pc. Furthermore, Ar–H, aliphatic C–H, C=N and C–O–C vibration bands of all the synthesized phthalocyanine compounds were observed at expected frequencies (Fig. S7, S10, S13 using compound 3bH₂Pc, Zn3b and In3b, as examples, respectively).

As shown in the ¹H-NMR spectrum of compound 3a, the signal corresponding to the HC=N group was observed at 8.57 ppm. Although 3a shows a proton signal of aromatic –OH at 9.82 ppm, this peak disappeared with the synthesis of 3b. The obtained proton signals of the HC=N group were seen at 8.6 ppm for 3bH₂Pc and 8.58 ppm for In3b. Also, aromatic –CH and proton signals of HC=N for Zn3b were obtained at 8.8–6.8 ppm. Aliphatic proton signals were found at 3.84 for 3bH₂Pc, Zn3b and In3b (Fig. S2, S4, S9 and S12 using compounds 3a, 3b, Zn3b and In3b as an example, respectively). In the mass spectra, molecular ion peaks were observed at 227.088 [M]⁺, 353.02 [M]⁺, 1415.20 [M]⁺, 1478.944 [M]⁺ and 1526.35 [M − Cl − 2H]⁺ for 3a, 3b, 3bH₂Pc, Zn3b and In3b, respectively (Fig. S1, S3, S6, S8 and S11 using compound 3a, 3b, 3bH₂Pc, Zn3b and In3b as an example, respectively).

3.2. Ground state electronic absorption spectra

Phthalocyanines exhibit two typical major bands, which are based on electronic transitions, like n–π* and π–π* transitions. Metallophthalocyanines and dihydrogen phthalocyanines, also called metal-free phthalocyanines, have the D_4h and D_2h symmetry, respectively. Due to these different symmetries, when metal-free Pcs exhibit two characteristic Q (Q_x and Q_y) bands, metallophthalocyanines exhibit one strong Q band in the UV-vis spectra. The Q bands are observed at 703 and 674 nm for 3bH₂Pc, 682 nm for Zn3b and 696 nm for In3b in DMSO. Because of the substitution of 4-{4-[(E)-{[4-methoxyphenyl]methylidene}amino]phenoxy} moieties on the Pc ring, the Q bands of compounds 3bH₂Pc, Zn3b and In3b were red-shifted in contrast to the unsubstituted ZnPc (672 nm) in DMSO. The electronic absorption spectra of all synthesized phthalocyanines are given in Fig. 1.

Fig. 1 Electronic absorption spectra of the non-ionic phthalocyanine compounds (3bH₂Pc, Zn3b, and In3b) in DMSO.

Aggregation significantly influences the photodynamic activity of phthalocyanines, leading to strong π–π stacking interactions, which reduce light absorption efficiency, suppress excited-state lifetimes, and cause decreased singlet oxygen generation. Therefore, the aggregation behavior of the synthesized non-ionic phthalocyanines was determined by measuring the absorbance values of solutions prepared at different molarities in DMSO. These measurements indicated that the synthesized imine-substituted non-ionic phthalocyanines did not show any aggregation in DMSO. Moreover, the concentrations ranging from 1 × 10⁻⁵ to 5 × 10⁻⁵ M for 3bH₂Pc, 2 × 10⁻⁶ to 10 × 10⁻⁶ M for Zn3b and 2 × 10⁻⁵ to 6 × 10⁻⁵ M for In3b in DMSO obey the Lambert-Beer law (Fig. 2 and Fig. S14 in the SI).

Fig. 2 Absorbance changes of Zn3b in DMSO at different concentrations ranging from 2 × 10⁻⁶ to 10 × 10⁻⁶ M.

3.3. Photophysicochemical yields

3.3.1. Fluorescence spectra and quantum yields. The fluorescence spectra of imine-substituted non-ionic phthalocyanines (3bH₂Pc, Zn3b, and In3b) were investigated in DMSO. The excitation spectra of all synthesized metal-free and metallophthalocyanines were similar to the absorption spectra and both spectra were mirror images of the emission spectra. The fluorescence emission maxima were determined at 713 nm for 3bH₂Pc, 695 nm for Zn3b and 712 nm for In3b. The observed data from absorption, emission and excitation spectra are listed in Table 1 (Fig. 1 and 3).

Fig. 3 Emission spectra of the 3bH₂Pc, Zn3b and In3b compounds in DMSO.

Table 1 Absorption, excitation and emission spectral data for the non-ionic phthalocyanine compounds in DMSO

Compound	Q band λmax (nm)	log ε	Excitation λEx (nm)	Emission λEm (nm)	Stokes shift
3bH2Pc	703 and 674	3.56 and 3.61	705	713	10
Zn3b	682	4.80	684	695	13
In3b	696	4.16	705	712	16

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The fluorescence quantum yields (Φ_F) of imine-substituted phthalocyanines are listed in Table 2. The Φ_F values (10⁻²) of 3bH₂Pc, Zn3b and In3b were determined as 1.7, 12.2 and 0.52, respectively. When compared with the Φ_F value of unsubstituted ZnPc (0.18), all synthesized imine-substituted phthalocyanines have lower Φ_F values in DMSO. Also, compound In3b has the lowest Φ_F value because of the heavy atom effect of the indium ion. Compared with amino-substituted structures,^30–33 the presence of methoxy substituents is not sufficient to activate the photoinduced electron transfer (PET) process. Consequently, methoxy-substituted 3bH₂Pc, Zn3b and In3b exhibit higher fluorescence quantum yields.

Table 2 Fluorescence, singlet oxygen and photodegradation quantum yields of the non-ionic phthalocyanine compounds in DMSO

Compound	ΦF (10^−2) DMSO	Φd (10^−4) DMSO	ΦΔ DMSO
3bH2Pc	1.7	0.7	0.12
Zn3b	12.2	28.0	0.71
In3b	0.52	3.0	0.85

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3.3.2. Singlet oxygen quantum yields. Singlet oxygen, which destroys cancerous tissue as a result of its reaction with biomolecules, is one of the most important parameters examined in determining the effectiveness of PDT. Singlet oxygen generation is strongly influenced by both solvent properties and structural features of the photosensitizer. The imine functionality is further substituted with a methoxy (–OCH₃) group, which is known to act as an electron-donating substituent through resonance. The presence of the methoxy group increases the electron density on the imine moiety and contributes to the extension of the π-conjugated framework of the phthalocyanine. This increased electron density may facilitate charge transfer within the excited state, which can enhance ISC processes.^34,35 Solvent polarity also plays a critical role in modulating the ISC. Polar solvents may facilitate ISC and enhance triplet state population, thereby improving singlet oxygen quantum yields.^19,36,37

In the present study, photophysicochemical measurements were performed in DMSO, a highly polar aprotic solvent known for its strong solvation ability and relatively high oxygen solubility. Moreover, as an aprotic solvent, DMSO does not form strong hydrogen-bonding interactions with the macrocycle, thereby minimizing specific solvent–solute interactions that could otherwise alter the intrinsic electronic properties of the phthalocyanine core. The solution, with non-ionic phthalocyanines containing 1,3-diphenylisobenzofuran (DPBF) as a quencher in DMSO, was irradiated with light every 5 s. The change in the absorbance of DPBF (417 nm) was monitored using the UV-vis spectra. The obtained spectra for Schiff base-decorated metal-free and metallophthalocyanines are given in Fig. 4 and Fig. S15, and the values are listed in Table 2. The Φ_Δ values were calculated as 0.12 for 3bH₂Pc, 0.71 for Zn3b and 0.85 for In3b in DMSO. The metal-free 3bH₂Pc exhibits a lower Φ_Δ value than the unsubstituted ZnPc (0.67) in DMSO, whereas In3b has the highest Φ_Δ value among all the investigated phthalocyanines. These enhancements arise from the strong heavy atom effect³⁸ of the indium ion,³⁹ which significantly facilitates intersystem crossing (ISC) and consequently singlet oxygen generation. In recent studies, the synthesized non-ionic phthalocyanines have similar Φ_Δ values compared with the other studied metal-free and metallophthalocyanines containing imine groups in DMSO.^28,40,41 Also, several studies have demonstrated that the incorporation of imine functional groups into π-conjugated systems can positively influence ISC processes and consequently, enhance singlet oxygen quantum yields. The presence of the imine linkage contributes to the extension and modulation of the conjugated electronic framework, altering the distribution of frontier molecular orbitals and facilitating more efficient population of the triplet excited state. In addition, the lone pair electrons on the nitrogen atom may participate in n → π* transitions, which are known to promote spin–orbit coupling and improve ISC efficiency.^28,42,43

Fig. 4 Typical spectrum for the determination of singlet oxygen quantum yield of the In3b compound in DMSO using DPBF as a singlet oxygen quencher.

3.3.3. Photodegradation quantum yields. Photodegradation studies, which yield necessary parameters for maintaining the activity of phthalocyanine and keeping the drug concentration constant, were conducted for determining the stability of phthalocyanines under light irradiation. Stable compounds have photodegradation quantum yields (Φ_d) in the order of 10⁻³ to 10⁻⁶, as reported in the literature.^44–46 Fig. 5 and Fig. S16 in the SI display the Φ_d spectra of imine-substituted phthalocyanines. The Φ_d values calculated from the spectra given in Fig. 5 and Fig. S16 in the SI are listed in Table 2. The Φ_d values of all synthesized imine substituted non-ionic phthalocyanines indicate in the order of 10⁻³ in DMSO. These results show that compounds 3bH₂Pc, Zn3b and In3b have sufficient stability. Also, Zn3b is the most unstable compound in DMSO.

Fig. 5 Typical spectrum for the determination of photodegradation of the Zn3b compound in DMSO.

3.4. In vitro studies

3.4.1. Cytotoxicity assessment. Based on the MTT assay results, a concentration of 5 μM was identified as the highest concentration that did not induce a statistically significant cytotoxic effect compared with the control group for Zn3b, In3b, and 3bH₂Pc. Therefore, this concentration was selected as the treatment dose for subsequent experiments (Fig. 6).

Fig. 6 Dose-dependent MTT cytotoxicity analysis of the phthalocyanine derivatives in PC3 cells: (A) 3bH₂Pc, (B) In3b, and (C) Zn3b. Data are presented as column graphs. Statistically significant differences compared with the control group are indicated by * (p < 0.05).

3.4.2. Effects of PDT on cell viability and apoptosis. Following PDT, cell viability decreased to 83.17% ± SEM in the 3bH₂Pc-mediated PDT group, 38.83% ± SEM in the In3b-mediated PDT group, and 48.48% ± SEM in the Zn3b-mediated PDT group. All phthalocyanine-mediated PDT treatments resulted in a statistically significant reduction in cell viability compared with the control group (p < 0.05) (Fig. 7).

Fig. 7 MTT assay results following PDT in PC3 cells: (A) 3bH₂Pc-mediated PDT, (B) In3b-mediated PDT, and (C) Zn3b-mediated PDT. The phthalocyanine derivatives were applied at a concentration of 5 μM. Data are presented as column graphs. Statistically significant differences compared with the control group are indicated by * (p < 0.05).

PDT mediated by different phthalocyanine derivatives resulted in varying levels of total apoptosis in PC3 cells. Following the 3bH₂Pc-mediated PDT, total apoptosis was observed at 5.08%, whereas In3b-mediated PDT induced a marked increase in total apoptosis to 46.27%, and Zn3b-mediated PDT resulted in 40.20% total apoptosis. All phthalocyanine-mediated PDT treatments led to a statistically significant increase in total apoptosis compared with the control group (p < 0.05). The apoptosis results are presented in Fig. 8.

Fig. 8 Apoptosis analysis following PDT in PC3 cells. (A) Representative flow cytometry dot plots showing Annexin V-FITC/PI staining after phthalocyanine-mediated PDT. (B) Quantitative analysis of the total apoptosis. Data are presented as column graphs and expressed as mean ± SEM (n = 3). Statistically significant differences compared with the control group are indicated by * (p < 0.05).

3.4.3. Effect of phthalocyanine-mediated PDT on cellular ROS levels. The DHE-based flow cytometric analysis revealed a marked increase in intracellular ROS levels in PC3 cells following phthalocyanine-mediated PDT (Fig. 9).

Fig. 9 Phthalocyanine-mediated PDT-induced intracellular ROS generation in PC3 cells. (A) Representative flow cytometry fluorescence histograms showing rightward shifts in the DHE fluorescence intensity (x-axis shift) following photodynamic therapy (PDT). (B) Quantitative analysis of ROS-positive cells (%), presented as column graphs. Data are expressed as mean ± SEM. Statistically significant differences compared with the control group are indicated by * (p < 0.05). tert-Butyl hydroperoxide (tBHP) was used as a positive control.

Quantitative evaluation demonstrated that 3bH₂Pc-mediated PDT resulted in a modest elevation of ROS-positive cells to 13.26%, whereas a pronounced increase was observed in cells treated with In3b-mediated PDT (49.31%) and Zn3b-mediated PDT (33.87%).

Consistent with the cytotoxicity and apoptosis data, metallophthalocyanines, particularly In3b, induced substantially higher ROS levels than the metal-free derivative, highlighting the critical role of the central metal ion in enhancing PDT efficacy. The elevated ROS production correlated well with the observed reductions in cell viability and enhancements in total apoptosis, supporting a ROS-mediated mechanism of cell death in PC3 cells following PDT.

In this study, the photodynamic effects of the imine-substituted metal-free and metallophthalocyanines (3bH₂Pc, Zn3b, and In3b) were further evaluated through in vitro biological assays. Although the Φ_d values of all Schiff base-substituted phthalocyanines were reported to be comparable, the presence of a central metal ion was shown to significantly enhance Φ_Δ, particularly in the In3b derivative due to the heavy atom effect. This photophysical advantage was clearly reflected in the biological outcomes observed in the present study.

Consistent with their favorable photophysical properties, all phthalocyanine derivatives exhibited minimal dark cytotoxicity at the selected concentration of 5 μM, confirming their suitability as photosensitizers for PDT. Upon light irradiation, a significant reduction in cell viability was observed in all phthalocyanine-mediated PDT groups, indicating effective photodynamic activation. Notably, In3b- and Zn3b-mediated PDT resulted in a markedly stronger decrease in cell viability compared with 3bH₂Pc-mediated PDT, which correlates well with the enhanced singlet oxygen generation associated with the presence of metal ions in the phthalocyanine core.^47,48

The analysis of apoptosis data further supported these findings, revealing a pronounced increase in total apoptotic cell death following In3b- and Zn3b-mediated PDT. The significantly higher apoptosis rates induced by these metallophthalocyanines suggest that metal-assisted enhancement of intersystem crossing and singlet oxygen production plays a critical role in triggering programmed cell death pathways.⁴⁹ In contrast, the relatively lower apoptotic effect observed for 3bH₂Pc may be attributed to the absence of a central metal ion, resulting in reduced ROS generation despite comparable Φ_d values.⁵⁰

4. Conclusions

In this work, novel imine-substituted metal-free and metallophthalocyanines were successfully synthesized and characterized using various spectroscopic methods. The photophysicochemical properties of compounds 3bH₂Pc, Zn3b and In3b were investigated in DMSO. Also, the effect of the central metal ion in the phthalocyanine ring on these properties was investigated. These measurements indicate that the Φ_d values of all synthesized Schiff base-substituted phthalocyanines are almost the same. In addition, it was observed that the presence of metal ions increased the singlet oxygen quantum yield. The combination of a heavy In atom and the substituent containing a conjugated Schiff base group increased the Φ_Δ value as expected. Inhibition of PET from the methoxy moiety to the Pc ring also increased the Φ_Δ value. The obtained results show that the newly synthesized imine-substituted metallophthalocyanines are efficient and convenient photosensitizers for PDT applications.

In conclusion, the present in vitro study demonstrated that imine-substituted metal-free and metallophthalocyanines exhibit distinct photodynamic activities depending on the presence of a central metal ion. All phthalocyanine derivatives showed negligible dark cytotoxicity at the selected concentration, confirming their biocompatibility under non-irradiated conditions. Upon light activation, phthalocyanine-mediated PDT significantly reduced cell viability and induced apoptotic cell death in PC3 cells.

This photodynamic effect was accompanied by a significant increase in intracellular ROS generation, with metallophthalocyanines, particularly In3b, inducing markedly higher ROS levels than the metal-free derivative. The enhanced ROS production correlated well with the observed reductions in cell viability and enhancements in total apoptosis, indicating that ROS-mediated oxidative stress plays a central role in the cytotoxic mechanism of phthalocyanine-mediated PDT.

These findings provide a strong foundation for further mechanistic investigations and future in vivo studies aimed at advancing phthalocyanine-based PDT strategies.

Conflicts of interest

There are no conflict of interest.

Data availability

The data supporting the findings of this study are available within the article and its supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6dt00397d.

Acknowledgements

This research has been supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK) via project number 219Z084.

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