Peripherally Crowded Cationic Phthalocyanines as Efficient Photosensitizers for Photodynamic Therapy

Abstract

Photodynamic therapy is a treatment modality of cancer based on the production of cytotoxic species upon the light activation of photosensitizers. Zinc phthalocyanine photosensitizers bearing four or eight bulky 2,6-di(pyridin-3-yl)phenoxy substituents were synthesized, and pyridyl moieties were methylated. The quaternized derivatives did not aggregate at all in water and retained their good photophysical properties. High photodynamic activity of these phthalocyanines was demonstrated on HeLa, MCF-7, and EA.hy926 cells with a very low EC₅₀ of 50 nM (for the MCF-7 cell line) upon light activation while maintaining low toxicity in the dark (TC₅₀ ≈ 600 μM), giving thus good phototherapeutic indexes (TC₅₀/EC₅₀) above 1400. The compounds localized primarily in the lysosomes, leading to their rupture after light activation. This induced an apoptotic cell death pathway with secondary necrosis because of extensive and swift damage to the cells. This work demonstrates the importance of a bulky and rigid arrangement of peripheral substituents in the development of photosensitizers.

Cancer is the second leading cause of death globally, accounting for one in six deaths in 2018, and its burdens continue to grow worldwide.¹ The treatment of all cancer types has not yet been solved, making investigations in this area of extraordinary high interest. Photodynamic therapy (PDT) is a modern method of cancer treatment, alternative to surgery or radiation therapy. It is characterized by noninvasive character, the possibility of targeting, and no serious long-term side effects, which may occur during other cancer treatment modalities.²⁻⁵ PDT uses special drugs, i.e., photosensitizers (PSs), along with excitation light and ubiquitous molecular oxygen to generate singlet oxygen that kills the cancer cells.

Previous studies have shown the great potential of phthalocyanines (Pcs) to become optimal PSs and revealed the basic structure–activity relationships.⁶⁻⁹ Upon light activation, Pcs are able to transfer energy to molecular oxygen, producing toxic singlet oxygen. They typically absorb light in the optical window of tissues (i.e., 650–850 nm), with high extinction coefficients of over 100 000 M^–1cm^–1. Such light penetrates deeply, which contrasts other well-known PSs, e.g., porphyrins, whose main absorption band (Soret band) lies typically at lower wavelengths (∼400 nm). Bulky substituents on the Pcs’ periphery are necessary to hinder undesirable aggregation, which would otherwise lead to the loss of photodynamic properties. However, this may not be sufficient in aqueous media, where the π–π interactions between planar and strongly hydrophobic Pc cores are extraordinarily strong. Various surfactants^10,11 or delivery systems¹²⁻¹⁵ have been reported to solve this obstacle, yet these systems may render in vitro evaluation and practical use difficult. From this point of view, the introduction of bulky charged substituents that suppress aggregation by repulsion forces is the most promising strategy.¹⁶⁻²⁰ Among charged species, cationic Pcs have been recently demonstrated to possess better in vitro PDT efficiencies than the anionic Pcs.^21,22 This fact was explained by an effect of environmental pH in cell compartments on Pcs’ ionization, by their different binding to serum proteins, interaction with biomembranes, subcellular localization, and relocalization after irradiation.

The motivation of this project was to evaluate how the rigidity of peripheral patterns, their number, and charge state (neutral or quaternized) are fundamental for achieving a high PDT efficiency of Pcs. For this purpose, the peripherally crowded Pcs 1, 1Me, 2, and 2Me containing 8 or 16 pyridyl moieties (neutral or quaternized) were designed (Chart 1), synthesized, and described from the photophysical point of view, and their photodynamic properties were evaluated in detail through in vitro studies. Zinc(II) as a central metal ion was chosen for its known good coordination stability in the Pc core, its straightforward synthetic availability, and, especially, its increasing singlet oxygen production based on the heavy atom effect.^6,7,23−25 Moreover, the selection of compounds was based on the recent positive preliminary results with 2Me, which was shown to be highly photoactive in the production of reactive oxygen species (ROS) after immobilization on tobacco mosaic virus (TMV) protein crystals, for use as heterogeneous catalysts in continuous-flow oxidation processes.²⁶

Chart 1. Structures of the Target Pcs.

The preparation of tetrasubstituted Pc 1 started with the synthesis of 4-(2,6-di(pyridin-3-yl)phenoxy)phthalonitrile, using (2,6-di(pyridin-3-yl)phenol)²⁶ and 4-nitrophthalonitrile as precursors, obtaining the product in 71% yield. Cyclotetramerization of this phthalonitrile in pentan-1-ol and with DBU as a base, at 140 °C, afforded Pc 1 in 6% yield, which was purified by column chromatography using a mixture of CHCl₃/MeOH (10:0.5) with 1% of pyridine, followed by size exclusion chromatography in Bio-Beads using DCM as an eluent. Alkylation of the pyridyloxy groups using methyl iodide in DMF afforded Pc 1Me in 82% yield (Scheme 1). Pcs 2 and 2Me were prepared using a previously described procedure.²⁶ All products were fully characterized by NMR, mass spectrometry, IR, and UV–vis. Importantly, the correlation of intensities between aromatic protons and the N-methyl groups in the ¹H NMR spectrum, together with a clear peak for the molecular ion in the HR-MS spectrum, confirmed the full quaternization of the pyridine moieties in Pcs 1Me and 2Me. The purity of the samples was assessed by HPLC and was found to be over 95% for all samples. Additionally, three peaks of positional isomers with different Q-band shapes of absorption spectra were detected during HPLC analysis of 1 (Figure S8), with abundances of 10, 77, and 11% (the middle peak most likely contained two isomers). However, assignment of the peaks to individual isomers was not possible. The isomers were not separated in 1Me, but a small difference in the absorption spectra in the ascending, top, and descending parts of the chromatographic peak indicated their presence.

Scheme 1. Synthesis of Pcs 1 and 1Me.

(i) K₂CO₃, DMSO, 18 h, 90°C, 71%; (ii) Zn(OAc)₂, DBU, pentan-1-ol, 48 h, 140°C, 6%; (iii) CH₃I, DMF, 12 h, 120°C, 82%.

All the prepared Pcs showed absorption spectra in DMF that are typical for monomeric Pcs, with sharp Q-bands over 680 nm and B-bands at approximately 360 nm (Table 1 and Figure 1). Evidently, quaternization did not affect the position and the shape of the Q-bands. Negligible splitting in the Q-band of 1 was the result of the presence of positional isomers, since these Pcs were prepared from monosubstituted phthalonitrile. The splitting was not observed for 1Me after quaternization. The absorption spectra of nonquaternized Pcs recorded in phosphate-buffered saline (PBS, the same results were obtained in water) differed significantly from those in DMF. A significant decrease of the Q-band intensity and appearance of a new blueshifted band of 1 indicated strong aggregation of this Pc under aqueous conditions (Figure 1a). Although the sharp Q-band of Pc 2, with only decreased intensity and slight broadening, suggested its presence in the monomeric form, no fluorescence was emitted from the PBS or water solutions (Figure 1c). Similar observations have been reported for extremely bulky phenoxy-substituted Pcs and were ascribed to the formation of atypical aggregates that do not differ from monomers in the shape of the absorption spectrum.^20,27,28 The absorption spectra of quaternized Pcs 1Me and 2Me in PBS, in turn, were also characteristic of the monomeric form and perfectly overlapped the spectra in DMF. In contrast to Pc 2, a strong fluorescence emission was preserved this case. The fully monomeric characters of 1Me and 2Me in water, which are a crucial prerequisite for obtaining good PDT properties in vitro, were further proven spectroscopically based on the accordance of the shape of excitation fluorescence spectra with the corresponding absorption spectra (Figure S13). The shape of the absorption spectra did not change, even at a concentration of 100 μM (Figure S13), indicating a very efficient inhibition of Pc aggregation by this extremely rigid arrangement of quaternized peripheral substituents.

Table 1. Photophysical Properties of Studied Compounds^a.

cpd.	solvent	λA, nm	λF, nm	ΦF (absol/comp)	τF, ns	ΦΔ (phos/chem)	τΔ, μs
1	DMF	682	689	0.29/0.25	2.89	0.60/0.59	17.7
1Me	DMF	684	692	0.18/0.17	2.19	0.57/0.51	15.2
	PBS	684	695	0.17/0.19	2.83	-	-
2	DMF	681	687	0.29/0.28	3.15	0.59/0.56	18.7
2Me	DMF	693	701	0.16/0.18	2.17	0.58/0.49	17.4
	PBS	690	700	0.16/0.16	2.34	-	-

^aAbsorption maximum (λ_A); emission maximum (λ_F); fluorescence quantum yield (Φ_F) determined either by the absolute method (absol) or comparative method (comp) (for details, see the Supporting Information); singlet oxygen quantum yield (Φ_Δ) determined either by the phosphorescence method (phos) or by the chemical method (chem) (for details, see the Supporting Information); fluorescence lifetime (τ_F); singlet oxygen lifetime (τ_Δ). Fluorescence lifetimes (τ_F) were determined using laser with λ_exc = 653.9 nm.

Figure 1.

Absorption spectra of compounds 1 (a), 1Me (b), 2 (c), and 2Me (d) in water (red), PBS (black), and DMF (blue) at a 1 μM concentration. The solutions of 1 and 2 were diluted from 100 μM DMSO stock solution. The inset in figure (c) shows the fluorescence of the DMF (left) and PBS (right) solutions (1 μM) of compound 2 (λ_exc = 366 nm).

Photophysical properties, especially quantum yields of singlet oxygen production (Φ_Δ), can suggest the impact of the studied Pcs for PDT. Fluorescence emission defined by fluorescence quantum yields (Φ_F) may be used for bioimaging and to localize PSs at the subcellular level or in the tissues. Two different methods have been employed for the determination of both Φ_Δ and Φ_F values in DMF to confirm the accuracy of the obtained data. DMF was a solvent of choice, because it ensures good solubility of the whole series, guarantees the monomeric character of compounds, and provides a relevant published value of Φ_Δ of unsubstituted zinc phthalocyanine, used as a reference in the chemical method (see below). The data are summarized in Table 1. The Φ_Δ values were determined with unsubstituted zinc Pc as the reference, using either the chemical method with 1,3-diphenylisobenzofuran as a selective scavenger of singlet oxygen or by the phosphorescence method observing phosphorescence of generated singlet oxygen at 1276 nm (Figure S12). The value of the singlet oxygen lifetime (τ_Δ) was about 17 μs for all the samples, which is in accordance with the data published in the literature.²⁹ All prepared Pcs were characterized by Φ_Δ values of 0.49–0.60 in DMF, which is in good agreement with the data previously published for similar zinc Pcs,^19,20 confirming their monomeric character and indicating the great potential of these derivatives for photodynamic treatments. For the determination of the Φ_F value (in DMF and PBS), an absolute method using an integrating sphere and a relative method using zinc Pc as the reference were used. In addition, in this case, the obtained Φ_F values corresponded each other, thus proving the relevance of the measured data. Interestingly, the nonquaternized compounds 1 and 2 possessed a higher Φ_F in DMF, close to 0.30, whereas Φ_F values of the corresponding quaternized analogues 1Me and 2Me were in the range of 0.16–0.19. The identical values of Φ_F for 1Me and 2Me were also determined in PBS, further confirming the monomeric character of 1Me and 2Me under aqueous conditions and making the following in vitro experiments reasonable. Fluorescence lifetimes (τ_F) ranged from 2.17–3.15 ns and correlated well with Φ_F values. For 1 and 2, in turn, no fluorescence was detected in PBS due to aggregation.

All four prepared compounds were subsequently evaluated in vitro on the various cell lines—human cervical carcinoma (HeLa), human breast carcinoma (MCF-7), nonmalignant mouse fibroblasts (3T3), and immortalized human endothelial cells (EA.hy926). Their photodynamic activity after activation by light (λ > 570 nm, 11.2 J cm^–2) was expressed as the half-maximal effective concentration (EC₅₀), while the inherent toxicity without activation (dark toxicity) was expressed as the half-maximal toxic concentration (TC₅₀); see Figures 2 and S17. The nonquaternized derivatives 1 and 2 precipitated above a 1 μM concentration in the cell-culture medium without any detectable toxicity toward HeLa cells either in the dark or upon activation. This is a consequence of the above-discussed tendency toward aggregation in water-based media, leading to inactive forms of PSs, for which these compounds were not evaluated further. Conversely, both quaternized Pcs 1Me and 2Me induced concentration-dependent toxicity upon light activation on all cell lines (Table 2). Both Pcs appeared to be most active against MCF-7 cells with EC₅₀ values of ∼0.049 μM. Approximately an order of magnitude lower activity was observed against HeLa cells. The results of both Pcs on nonmalignant 3T3 cells differed substantially. While high toxicity was determined for 2Me, statistically significant lower phototoxicity was observed for 1Me in comparison with MCF-7 cells (p = 0.049, Welch’s t test). This suggests that 1Me may have a beneficial effect in the PDT treatment, and even after light activation, it may efficiently destroy some types of carcinoma cells while preserving surrounding healthy tissues to some degree. Nevertheless, the results do not exclude 2Me from further studies, since the selectivity in PDT is primarily driven by irradiation of the target areas only. Moreover, this compound exerted much better photoactivity than 1Me independently of the type of cell line. In comparison with clinically approved PS, both studied compounds appeared to be very efficient; their activity was much higher than for the sulfonated hydroxyaluminum Pc (S₃AlOHPc) that has been approved in clinical practice in Russia under the trade name Photosens. EC₅₀ values determined for S₃AlOHPc by our research group under the identical experimental conditions were higher than 2 μM for all cell lines tested (see Table 2). Photodynamic treatment of HeLa cells using both investigated compounds without any previous incubation, i.e., irradiation, started immediately after the application of dyes (Figure S18), resulted in EC₅₀ values of 1.28 ± 0.68 and 0.178 ± 0.024 μM for 1Me and 2Me, respectively. Even though a decrease in activity was expected, both compounds were proven to maintain their photodynamic activity well. This preservation of photodynamic activity may be utilized in PDT modalities where omission of a PS’s uptake (or its ample reduction) is conveniently availed (e.g., in vascular-targeted photodynamic therapy). Therefore, a similar experiment utilizing human endothelial cells (EA.hy926) was conducted, with comparable results (Table 2). The dark toxicities of both quaternized compounds were low (TC₅₀ > 600 μM, Figures 2 and S17 and Table 2) and were either comparable or even lower than those of similar hydrophilic multicationic compounds.²¹ Phototherapeutic indicies (i.e., TC₅₀/EC₅₀ ratios) revealed very favorable values (HeLa cells) of 1409 and 5691 for 1Me and 2Me, respectively.

Figure 2.

(a) Photodynamic activity of compound 1Me on 3T3 (red), HeLa (black), and MCF-7 (blue) cells (λ > 570 nm, 12.4 mW cm^–2, 15 min, 11.2 J cm^–2) and dark toxicity of 1Me on HeLa cells (green). (b) Subcellular localization of 1Me (red) in HeLa cells. Cells were stained for mitochondria (green) and lysosomes (blue) by organelle-specific fluorescent probes. (c) The intensity profiles of 1Me (red), lysosomes (blue), and mitochondria (green) alongside the white bar shown in part (b).

Table 2. Comparison of Uptake, Dark Toxicity (TC₅₀ Values), and Photodynamic Activity (EC₅₀ Values) of Studied Pcs in Different Cell Lines^a.

		EC50 (μM)					TC50 (μM)	TC50/EC50
cpd.	uptake (nmol per mg of protein), HeLa	HeLa	MCF-7	3T3	EA.hy926e	HeLae	HeLa	HeLa
1	-	>1b	-	-	-		>1b	-
1Me	0.26	0.48 ± 0.25	0.050 ± 0.014	2.12 ± 1.4	3.43 ± 0.61	1.28 ± 0.68	675 ± 17	1409
2	-	>1b	-	-	-		>1b	-
2Me	0.50	0.11 ± 0.027	0.047 ± 0.016	0.068 ± 0.029	0.50 ± 0.07	0.18 ± 0.024	601 ± 6	5691
S3AlOHPc		2.07 ± 0.29c	2.04 ± 0.31d	2.37 ± 0.63d	-		>1500d	>725

^aData are presented as the TC₅₀ or EC₅₀ values ± standard deviations. TC₅₀/EC₅₀ refers to the phototherapeutic index. Irradiation conditions: λ > 570 nm, 12.4 mW cm^–2, 15 min, 11.2 J cm^–2. At least three independent experiments each in quadruplicate were performed.

^bThe compounds precipitated above this concentration.

^cData from ref (31).

^dData from ref (21).

^eWithout preincubation. The cells were irradiated immediately after application of Pc.

Active Pcs 1Me and 2Me were further studied in detail on HeLa cells. Their uptake after 12 h of incubation reached values of 0.26 and 0.50 nmol per mg of protein, which are comparable with those of other multicationic Pcs.²¹ Uptake of quaternized compounds led to their accumulation in the endolysosomal compartment of HeLa cells, as confirmed by fluorescence microscopy (Figures 2b,c, S15 and S16). Unfortunately, both compounds photobleached quickly in the cells. Together with rather rapid relocalization to the cytoplasm, these aspects made 1Me and 2Me difficult to image in their primary subcellular localization (endosomes and lysosomes). The entire rapid process of photoactivation with a violent burst-like release from lysosomes accompanied by further successive photobleaching is shown in the instructive video of irradiation of HeLa cells treated with 1Me (Supporting Information). This boisterous relocalization process with simultaneous oxidative damage to cellular components can lead to cell death. With lysosomes as the primary site of photodynamic damage, irradiation of the cells leads to activation of extrinsic- and intrinsic-pathway initiator caspases 8 and 9, respectively, and executioner caspases 3/7 at the same time, presumably by lysosomal enzyme cleavage of pro-caspases (Figures 3a and S19). Using annexin V luciferase fusion proteins and a DNA-binding probe allowed for real-time monitoring of cell demise. Exposure of phosphatidylserine, monitored as an increase of the luminescence signal, began readily after irradiation, followed by the increase in fluorescence after an apparent lag as a result of the subsequent loss of membrane integrity during secondary necrosis (Figures 3b–e and S20). This lag between the inception of the increase in both signals can be used to distinguish between apoptosis followed by secondary necrosis (observed as pronounced delay) and primary necrosis (observed as short or absent delay between an increase in luminescence and fluorescence signals).³⁰ A substantial delay (>5 h) is observable in apoptosis-positive controls (camptothecin and bortezomib as the inducers of intrinsic-pathway-mediated apoptosis (Figure S20) and FasL and TRAIL as the extrinsic-pathway inducers (Figures 3e and S20), while both studied compounds displayed evidence of secondary necrosis faster at higher dosages (even within 3.5 h after light activation). Hydrogen peroxide (which is known to induce oxidative damage and necrosis) resulted in initiation of the loss of membrane integrity within half an hour after addition to the cells, which is in accordance with the necrotic type of cell death. Digitonin (necrosis via cellular membrane permeabilization) induced devastating nonapoptotic cell death even more rapidly (Figure 3d). Cell death processes after light-induced photodynamic action of 1Me and 2Me involves the initial induction of apoptosis, but thanks to extensive and swift damage to the cells by a higher dosage of PSs, secondary necrosis might take over even within 3.5 h.

Figure 3.

Cell death determinations: (a) Caspases 3/7 (green), 8 (red), and 9 (blue) activities for 2Me in different times after irradiation. (b–e) Annexin V (green) and DNA-binding probe (red) binding profiles for 2Me, at EC₁₅ (b) and EC₈₅ (c), digitonin (d; necrosis control), and FasL (e; apoptosis control).

In conclusion, this work has demonstrated that an efficient rigid arrangement of peripheral cationic substituents on the aromatic core of Pcs efficiently inhibits aggregation, even in water. This keeps the PS molecules exclusively in a monomolecular state, which is very important for providing good photophysical properties and concomitantly high photodynamic activity. This outcome was demonstrated on cancerous cell lines for the cationic compounds 1Me and 2Me, providing much better EC₅₀ values than the clinically approved S₃AlOHPc. Further in vitro investigation revealed that the main cell demise pathway is through apoptosis with subsequent secondary necrosis after the rupture of the lysosomal vesicles, i.e., the organelles of primary subcellular localization. Therefore, the concept of introducing charged moieties on the rigid peripheral substituents was confirmed as a highly viable approach for delivering novel, highly active PSs.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00045.

• Synthesis, full characterization of target products, additional absorption spectra, NMR spectra, experimental parts (PDF)

• Photodynamic activity of 1Me on HeLa cells (MP4)

Author Contributions

^# M.H. and A.R. contributed equally.

The authors declare no competing financial interest.