Aggregation–disaggregation pattern of photodynamically active ZnPcS₄ and its interaction with DNA alkylating quinone: effect of micellar compactness and central metal ion

Abstract

The hypoxic nature of solid tumours and the aggregation of photosensitizers in physiological environments are two governing limiting factors in photodynamic therapy (PDT). This article describes the effect of sensitivity to polarity and the central metal ion on the aggregation–disaggregation pattern of the PDT-active metallophthalocyanine ZnPcS₄ and its relevant interaction with a DNA alkylating quinone in an aqueous medium, as well as in a biomimicking micellar microenvironment, under hypoxic conditions to overcome the limitations of PDT. Steady-state absorption and emission, time-resolved fluorescence (TRF), fluorescence anisotropy and dynamic light scattering (DLS) techniques were employed for this purpose. Similarly to AlPcS₄, the spectral behaviour of ZnPcS₄ remains immune to anionic SDS and also displays a significant interaction with the positively charged head group of cationic surfactants (DTAB, TTAB, and CTAB). In the premicellar region, surfactant-induced aggregation of the probe molecules is dominant. Above the critical micellar concentration (CMC), AlPcS₄ fully disaggregates into monomers but the tendency of ZnPcS₄ to undergo monomerization increases with a decline in the compactness of cationic micelles. The increase in the fluorescence anisotropy of ZnPcS₄ in a fully micellized system upon reducing the compactness of the micelles confirms deeper penetration of the dye inside the micelles, a behaviour which is the reverse of that of AlPcS₄. The latter remained resistant in neutral micelles, whereas ZnPcS₄ substantially monomerized on an increase in the TX-100 concentration and distributed itself at the shear surface and in the deeper hydrophobic core of a fully micellized system. Dye-induced formation of micellar clusters was identified by DLS measurements, especially in cationic micelles. The interaction of PDT-active ZnPcS₄ with DNA alkylating 2,5-dichlorodiaziridinyl-1,4-benzoquinone (AZDCIQ) was studied in an aqueous medium, as well as in all the micellar microenvironments under study, and found to be more prominent in cationic micelles compared with the other systems. However, the association constants (K_a) thus obtained in the cationic micelles were found to be greater for ZnPcS₄ than for AlPcS₄. Our findings may prove instrumental in expanding the effectiveness and applicability of metallophthalocyanines in terms of understanding their aggregation pattern and interaction with DNA alkylating quinones in a hypoxic micellar microenvironment.

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Introduction

Metallophthalocyanines are second-generation photosensitizers that have a structural resemblance to porphyrins and exhibit significant properties such as intense brightness, high thermal and chemical stability, and high efficiency in electron transfer.^1–4 These dyes are widely used in many fields such as optoelectronics,⁵ light-emitting devices,⁶ chemical sensors,⁷ and, most importantly, as photosensitizers in photodynamic therapy (PDT).⁸ In an aqueous medium, owing to the presence of a large hydrophobic macrocyclic skeleton, metallophthalocyanines have a tendency to stack to form dimers or larger aggregates.^9–11 Aggregated species are readily distinguished from their monomer counterparts by using UV-visible absorption spectroscopy, as they display an additional peak on the blue and/or red side of the monomer peak. This aggregation of metallophthalocyanines has a direct effect on their photophysical properties, rendering a normally active photosensitizer inactive via self-quenching and hence reducing its efficacy for the application of PDT.¹²

Photochemotherapy or PDT is a potential cancer treatment process that involves the irradiation of photosensitizers by light. Upon absorption of light, a photosensitizer forms a long-lived triplet excited state via intersystem crossing from a singlet excited state. In the triplet state, the photosensitizer can transfer an electron or energy to generate active molecular species such as free radicals (pathway I) and singlet oxygen (pathway II).^1,13,14 These short-lived species generated by both pathway I and II mechanisms in PDT are highly toxic in a biological environment such as a tumour cell.^15,16 Of the above-mentioned mechanisms (pathways I and II), pathway II is generally considered to be the most important process in PDT as it is directly involved in the killing of tumour cells. Hence, the presence of molecular oxygen in the target cell is the key to the success of PDT. Ironically, tumour cells are often hypoxic in nature; hence, the efficacy of killing tumour cells via the type II mechanism is limited.¹⁷ To overcome this limitation, Alegria et al. have proposed a new hypothesis, whereby a biologically reducible alkylating drug of which the redox potential is nearly equal to or more positive than that of oxygen is used.^1,2,16 According to this hypothesis, as photosensitizers can efficiently photoreduce oxygen they will also photoreduce a drug that has a redox potential nearly equal to or more positive than that of oxygen. If the drug used in this process is a DNA alkylating agent, then it will alkylate DNA after successful photoreduction, which results in cell death. Bioactive aziridinyl quinones are often used for the above-mentioned application, as they possess a photoreducible quinone moiety and an aziridinyl group, which, as a consequence of photoreduction, can form covalent bonds with a variety of cellular components including DNA.^1,2,18,19 To support the above hypothesis, a complete understanding of the physicochemical behaviour of PDT dyes in the absence and presence of an alkylating quinone in the cellular environment is necessary.

In the present article, the aggregation–disaggregation behaviour of zinc phthalocyanine and its interaction with a DNA alkylating quinone are studied in different micellar microenvironments, as micelles are commonly known as biomimicking agents because of their close resemblance to the structure of cell membranes.^20–22 The importance of membranes in biological systems lies in their capacity to provide a matrix for arranging reactions sequentially for efficient interaction. Substrate molecules may be sequestered and organized in the micellar interior or on the micellar boundary.^23–27 Micelles are also known for their action on such aggregating species, as they reduce the aggregation and increase the photoactivity of dyes. A change in surfactant concentration has a significant effect on the aggregation behaviour of macrocyclic dyes, which enables us to understand the aggregation pattern of dyes in biomimicking systems.^9,28,29 The idea behind choosing different surfactant systems is to provide a confined matrix that comprises differently charged regions with various polarities for the encapsulated PDT-active dye and DNA alkylating quinone, which will bring them together as it may result in an increase in the possibility of their association or alter their interaction pattern. Charges on micelles may also have a pronounced effect on such types of interactions.³⁰ The characterization of metallophthalocyanines in micellar systems has been reported in the literature by our group, as well as by others.^9,28,31,32 However, the main aim of this manuscript is not only to monitor the micelle-induced aggregation–disaggregation pattern of metallophthalocyanines but also, most importantly, to investigate the dye–quinone interaction inside the micellar microenvironment with simultaneous changes in micellar compactness and the central metal ion. To the best of our knowledge, the interaction of PDT-active ZnPcS₄ with a DNA alkylating quinone (AZDCIQ) in an aqueous medium, as well as biomimicking systems, along with the effects of the central metal ion and micellar compactness on the dye–quinone interaction in a hypoxic low-pH environment have not been reported so far. This information may be extremely useful for the responsiveness of the ZnPcS₄–quinone system in cell-like environments and emphasizes the novelty of the present article. Hence, a conspicuous attempt has been made to demonstrate the aggregation pattern of ZnPcS₄ and its interaction with 2,5-dichlorodiaziridinyl-1,4-benzoquinone (AZDCIQ) (Scheme 1) in neutral micelles, as well as in different cationic micelles of varying chain lengths, using UV-visible absorbance, steady-state fluorescence, fluorescence anisotropy, TRF spectroscopic studies and DLS techniques.

Scheme 1 Structures of (a) ZnPcS₄, (b) AlPcS₄, and (c) AZDCIQ.

Results and discussion

Interaction of metallophthalocyanines with different micellar systems

The absorption spectra of metallophthalocyanines exhibit a typical absorption band between 650 and 700 nm referred to as the Q band and a broader peak between 300 and 400 nm known as the Soret or B band.³³ The present investigations mainly focus on the photophysical properties of zinc phthalocyanine in different micellar microenvironments. The photophysical properties, as well as the related bioactivity, of a metallophthalocyanine are greatly influenced by its aggregation behaviour, which occurs due to interactions between the π systems of the macrocyclic rings. In a dimeric metallophthalocyanine, non-radiative deactivation by internal conversion from the S₁ state to the S₀ state is highly efficient and competes with the emission of photons (fluorescence) and intersystem crossing.¹² When these dimers are excited no appreciable singlet emission is detected; therefore, such aggregation must be avoided, and one way to circumvent the aggregation phenomenon is by incorporating the phthalocyanine into other self-aggregating systems such as micelles to understand the photodynamic behaviour of this macrocyclic system in a biomimicking environment. In this article, a conspicuous attempt has been made to analyze the rich photophysical behaviour of a metallophthalocyanine in differently charged micellar microenvironments and its relevant interaction with a DNA alkylating quinone. ZnPcS₄ displays significant variations in its absorption, as well as emission, spectra on the gradual addition of neutral (TX-100) and cationic surfactants (CTAB, DTAB, and TTAB). This change in the photophysical properties of ZnPcS₄ in neutral and cationic surfactants is probably because of hydrophobic and electrostatic interactions of ZnPcS₄ with the surfactant, which affect the aggregation–disaggregation behaviour and rate of monomerization of ZnPcS₄, whereas, in the case of the anionic surfactant SDS, the absorption and emission spectra of ZnPcS₄ remain unchanged, which is probably due to the repulsive interaction between the anionic dye and the anionic surfactant (Fig. S1†). Fluorescence anisotropy and TRF experiments also support the above facts, as discussed in the next section. All the dye–micelle interactions were monitored at a pH of 7.4, as well as a pH of 5.6 (excluding SDS), which shows similar results unless stated otherwise.

Surfactant-induced aggregation–disaggregation of ZnPcS₄

In water at physiological pH (7.4), ZnPcS₄ (1.897 × 10⁻⁶ M) exhibits two major bands, at 334 nm (the Soret or B band) and the Q band between 600 and 700 nm. The Q band mainly consists of three peaks at 583 nm, 634 nm and 674 nm. The band that appears at 583 nm is due to vibronic relaxation in the Q transition, whereas the 634 nm and low-energy 674 nm bands correspond to the dimer and monomer, respectively.^33,34 On the gradual addition of the cationic surfactant CTAB, an aqueous solution of ZnPcS₄ displays a remarkable change in the Q band of the absorption spectrum. Hence, a series of cationic surfactants of varying chain lengths was introduced here to study the peculiar behaviour of the macrocyclic dye (Fig. 1a and S2†). On the addition of a very low concentration of the cationic surfactant (for CTAB 0.01 mM, for TTAB 0.07 mM, and for DTAB 0.07 mM), the absorption intensity at 634 nm, which corresponds to the dimeric form of ZnPcS₄, significantly decreased, whereas the B band underwent bathochromism of 4 nm in its position. This decrease in absorption intensity may be attributed to a decrease in dimer concentration, which in turn may have increased the percentage of monomers or other more highly aggregated forms. On further addition of the cationic surfactant, a new peak in the red region at 679 nm gradually appeared and subsequently increased with an increase in the surfactant concentration. This newly generated peak exactly corresponds to the monomeric form of ZnPcS₄. The bathochromic shift in the absorption peak of the monomeric form compared with that in an aqueous medium is probably because of a change in the microenvironment due to the insertion of the dye in a micelle. TRF and fluorescence anisotropy measurements may reveal more about the micelle-induced monomerization and aggregation–disaggregation pattern in the micellar system, which is discussed later.

Fig. 1 Absorption (a) (i to viii: 0.0, 0.012, 0.025, 0.367, 0.729, 1.08, 2.32, and 3.65 mM) and emission (b) spectra of ZnPcS₄ with increasing concentrations of CTAB at 298 K. The inset shows the change in emission intensity with the increase in CTAB concentration.

Zinc phthalocyanine tetrasulphonate is a highly emissive phthalocyanine in an aqueous medium and its emission exclusively arises from its monomeric form. In an aqueous medium at room temperature, the emission spectrum of ZnPcS₄ displays a single unstructured band at 682 nm irrespective of the excitation wavelength. At lower concentrations of a cationic surfactant (CTAB, for instance), a dramatic decrease in the emission intensity of ZnPcS₄ is observed until a surfactant concentration of 0.025 mM is reached (Fig. 1b). This decrease in emission intensity could be because of the formation of non-fluorescent larger aggregates. However, on a further increase in the surfactant concentration the emission intensity starts to rise continuously and after micellization it becomes saturated. A plausible cause of this continuous rise in emission intensity could be due to an increase in the number of monomeric species, as is also observed in the absorbance spectra. However, the regeneration of monomers may be attributed to the surfactant-induced disaggregation of previously aggregated ZnPcS₄ molecules. The surfactant-induced aggregation of ZnPcS₄ at premicellar concentrations can be rationalized on the grounds of electrostatic interaction between the negatively charged dye and the positively charged surfactant monomer. This monomeric cationic surfactant attracts the negatively charged dye towards itself, which actually provides a matrix for the formation of larger aggregates owing to the predominant hydrophobic interactions between the phthalocyanine molecules. Larger aggregates of ZnPcS₄ are non-fluorescent species that ultimately result in a sudden decline in emission intensity. On further addition of the cationic surfactant, the electrostatic attraction between the surfactant and the dye overcomes the hydrophobic interactions between the phthalocyanine molecules, which results in the dissociation of larger aggregates into the highly emissive monomeric form and non-emissive dimeric species, which is reflected in an increase in the emission intensity. It is pertinent to mention here that the extent of the increase in the emission intensity of ZnPcS₄ varies among the different cationic surfactants being studied, because of differences in the extent of monomerization induced by altering the chain length of the surfactant (discussed in detail in the next section).

In the case of a non-ionic microenvironment, on the gradual addition of TX-100 to an aqueous solution of ZnPcS₄, the absorbance at 677 nm (which corresponds to monomeric species) continuously increases at the expense of the absorbance due to dimers (which occurs at 633 nm), as observed in Fig. 2a. This is corroborated by the increase in the emission intensity with the subsequent addition of TX-100. This disaggregation of the dimeric form in a TX-100 micelle is because of hydrophobic interactions between the dye and the surfactant. As well as the emission maxima, ZnPcS₄ also displays an emission shoulder near 696 nm. This shoulder at 696 nm appears immediately after the critical micelle concentration of TX-100 is reached (Fig. 2b), which is probably due to the regeneration of some monomer species deeper inside the micelle.

Fig. 2 Absorption (a) and emission (b) spectra of ZnPcS₄ with increasing concentrations of TX-100 (i to xxii) (0.0 to 3.0 mM) at 298 K.

Solvation dynamics of ZnPcS₄ in different micellar microenvironments

The tendency of this macrocyclic dye to undergo monomerization in both aqueous and micellar microenvironments was also investigated by TRF using the time-correlated single-photon counting (TCSPC) technique. In an aqueous solution, ZnPcS₄ (1.89 μM) exhibits a best-fitted biexponential fluorescence decay profile (Table 1), which predominantly corresponds to dimers (α₂ = 96.59%, τ₂ = 0.164 ns) and fewer monomers (α₁ = 3.41%, τ₁ = 2.89 ns). (The percentage contributions of both species depend on the concentration of the dye used in the experiment.) On the addition of a very low concentration of a cationic surfactant (CTAB, TTAB, or DTAB), the first interruption that is made in the microenvironment of the dye reduces this emission to a negligible value, as a consequence of which we were unable to record the decay profile of ZnPcS₄ at this particular surfactant concentration (Fig. 3a and S3†). On the basis of this behaviour, one could predict that this sudden fluorescence quenching effect is due to an aggregation phenomenon induced by the cationic surfactant in the pre-existing dimer and monomer of ZnPcS₄ in the aqueous solution, which forces them to assemble into larger aggregates. Such an abrupt change in ZnPcS₄ is also confirmed by the steady-state absorption and emission spectra (discussed earlier), where the dimer peak at 634 nm exhibits a decline in its absorption intensity and a subsequent reduction in fluorescence intensity upon the initial increase in the cationic surfactant concentration. On further addition of the cationic surfactant to the aqueous solution of the macrocyclic dye, larger aggregates dissociate into their monomeric and dimeric counterparts. After the CMC of all the cationic surfactants is reached, the percentage of the monomer rises from 3.41% to 8.48%, 13.31%, and 43.63% for CTAB, TTAB, and DTAB, respectively (Table 2). This is also reflected in the fluorescence decay curve, which rises again (Fig. 3b and S3†). As was true for the increase in the fluorescence intensity (Fig. 1b), an increase in the surfactant concentration leads to monomerization of the ZnPcS₄ dye, which contributes largely to its decay profile. The fact that the lifetime of ZnPcS₄ in a micellar system is comparatively slightly longer is probably because the dye locates itself in a less polar region (probably the Stern layer, owing to electrostatic attraction between the anionic dye and the cationic micelle) compared with that in the bulk aqueous phase.²⁸

Table 1 Fluorescence decay parameters of ZnPcS₄ in aqueous and micellar media at 298 K

Environment	τ1 (ns)	α1 (%)	τ2 (ns)	α2 (%)	τ3 (ns)	α3 (%)	τavg	χ^2
Water (682 nm)	2.89	3.41	0.164	96.59			0.256	1.14
SDS (14.0 mM, 682 nm)	2.94	3.58	0.143	96.42			0.243	1.21
CTAB (3.65 mM, 683 nm)	2.93	8.48	0.242	91.52			0.469	0.97
TX-100 (3.0 mM, 684 nm)	3.26	30.67	0.267	69.33			1.184	0.98
TX-100 (3.0 mM, 696 nm)	3.23	55.73	0.373	40.14	5.4	10.19	2.35	1.14

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Fig. 3 Fluorescence decay curves of ZnPcS₄ in DTAB at premicellar (a) and postmicellar (b) concentrations at 298 K.

Table 2 Fluorescence decay parameters of ZnPcS₄ in aqueous, CTAB, TTAB, and DTAB media at 298 K

Environment	τ1 (ns)	α1 (%)	τ2 (ns)	α2 (%)	τavg	χ^2
Water	2.89	3.41	0.164	96.59	0.256	1.14
CTAB (3.65 mM)	2.93	8.48	0.242	91.52	0.469	0.97
TTAB (12.73 mM)	3.19	13.31	0.260	86.69	0.649	0.89
DTAB (26.53 mM)	3.33	43.63	0.370	56.37	1.661	0.91

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As discussed earlier, the emission maximum of ZnPcS₄ in a neutral TX-100 micelle is observed at 684 nm. Above the critical micelle concentration of TX-100, the same emission spectrum develops a distinct hump at 696 nm. Therefore, in order to differentiate the excited-state characteristics of these two perceptible features in the emission band of the macrocyclic dye in a neutral micellar microenvironment, the decay profile of ZnPcS₄ was monitored at both 682 and 696 nm (Fig. 4). The fluorescence decay kinetics of ZnPcS₄ at 682 nm and 696 nm is described by bi- and tri-exponential fitting, respectively, and is summarised in Table 1. The decay profile of an aqueous solution of ZnPcS₄ monitored at 682 nm indicates that a long-lived monomer (τ₁ = 2.89 ns) is present as a minor contributor and a short-lived dimer (τ₂ = 0.164 ns) is present as a major contributor. As the concentration of TX-100 in the micelle continues to increase, the dimeric form of ZnPcS₄ disaggregates into monomers owing to hydrophobic interactions between the dye and the neutral surfactant and hence the percentage contribution of the monomer (τ₁) continuously rises from 3.41% to 30.67% (Fig. 4 and Table 1). Interestingly, it is also observed that the monomeric species displays little increase in its excited-state lifetime (2.89 to 3.26 ns), which is probably because of a decrease in micropolarity around the probe. Furthermore, the fluorescence decay profile of ZnPcS₄ when monitored at 696 nm in a fully micellized system is best fitted by a three-component model (Table 1). Here, in addition to the species with excited-state lifetimes of ∼0.3 ns and 3.26 ns, a new species with a slightly longer lifetime (5.4 ns) is observed. This can be attributed to redistribution of monomeric ZnPcS₄, where most of the monomeric form (with an excited-state lifetime of 3.26 ns) is present at the shear surface or in the palisade layer of the TX-100 micelle. However, some dye molecules further locate themselves in a deeper region, probably the apolar core region of the TX-100 micelle. This is also confirmed by the longer excited-state lifetime (5.4 ns) of the monomer in a more apolar region. The tendency of ZnPcS₄ to distribute itself in a nonpolar region is also supported by the hydrophobic nature of the dye. As the amount of micelles increases, this component with a longer lifetime becomes increasingly prominent. Thus, it may be concluded that the monomeric form (τ₁ and τ₃) of ZnPcS₄ is simultaneously distributed in two different regions of a neutral TX-100 micelle, at the shear surface or palisade and in the more hydrophobic core region.

Fig. 4 Fluorescence decay curves of ZnPcS₄ with increasing concentrations of TX-100 (i to v: 0.0, 0.1, 0.25, 1.39, and 3.0 mM) at 298 K. The inset shows the fluorescence decay of ZnPcS₄ monitored at 696 nm with increasing concentrations of TX-100 (i to iv: 0.0, 0.25, 1.39, and 3.0 mM) at 298 K.

Hence, the steady-state absorption/emission and TRF data for ZnPcS₄ in an aqueous medium, as well as in different micellar microenvironments, show that the aggregation–disaggregation pattern of ZnPcS₄ is strongly influenced by neutral and cationic micelles, whereas insignificant interaction occurs in the case of anionic SDS owing to repulsive interaction between the similarly charged species. In cationic micelles (CTAB, TTAB, and DTAB), both hydrophobic and electrostatic interactions come into play for a negatively charged fluorophore. Hence, owing to electrostatic attraction larger aggregates of ZnPcS₄ are observed in the premicellar region, which subsequently dissociate into monomers near the CMC and above. In neutral micelles, where only hydrophobic interactions are involved, the percentage of monomeric species continuously increases on the addition of a surfactant to an aqueous solution of ZnPcS₄. In a fully micellized TX-100 medium, the dye distributed itself in two different regions of the micelles, one near the shear surface, which corresponds to the ∼3.26 ns component, and the other in the deeper core region, which corresponds to the ∼ 5.4 ns component.

Identification of rotational confinement of ZnPcS₄ in a biomimicking microenvironment

The effect of a micellar microenvironment on the dynamic properties of ZnPcS₄ can be deduced from restrictions on its motion in that particular environment. In this regard, the steady-state fluorescence anisotropy technique is employed to provide a wealth of information about the microenvironment in the immediate vicinity of the fluorophore.³⁵ Fluorescence anisotropy is a sensitive indicator that offers a platform for understanding the physical characteristics and degree of rigidity imposed by confined micellar media around the probe. In an aqueous medium, the anisotropy value is quite low because of the free rotation of the fluorophore. If the fluorophore interacts strongly with micelles, then its anisotropy value should increase because micelles provide more confined media, which limits the free motion of the fluorophore to some extent. In an aqueous solution, the anisotropy value (r) of ZnPcS₄ was found to be ∼0.009. In the case of TX-100, the anisotropy value (r) of ZnPcS₄ increases with an increase in the surfactant concentration and, when all the molecules are fully micellized, it reaches a constant value of 0.048 (Fig. 5a). In the premicellar concentration region, with an increase in the surfactant concentration ZnPcS₄ displays very little increase in its value of r. On attaining a fully micellized condition at the CMC, the rotational diffusion of the dye decreases significantly, which is manifested as an increase in the value of r to 0.048. Upon a further increase in the surfactant concentration, the anisotropy value became stable at 0.05, which suggests that the microheterogeneous environment induces restrictions on the motion of the dye in a fully micellized medium. Thus, the anisotropy value of ZnPcS₄ in a TX-100 micelle strongly indicates that the fluorophore is located in a more confined environment, i.e., inside the micelle rather than in the outer bulk phase. In the case of cationic surfactants (CTAB, TTAB, and DTAB), the anisotropy value of ZnPcS₄ displays a quite different trend from that in neutral micelles. With an increasing, very low concentration of the cationic surfactants in the premicellar region, ZnPcS₄ exhibits an abrupt increase in the value of r (0.45) (Fig. 5b and S4†). This high anisotropy value is because of the presence of larger aggregates of the dye in this concentration range, as described earlier. On a further increase in the surfactant concentration, the macrocyclic dye disaggregates into monomers and dimers, which results in a sudden decrease in the values of r. In a fully micellized medium, the anisotropy values were found to be 0.025, 0.043, and 0.05 in CTAB, TTAB, and DTAB, respectively, which has the same explanation as in the case of TX-100 micelles. Hence, from the above observation it can be concluded that ZnPcS₄ is located in a more confined environment within the micelle rather than the exterior bulk aqueous phase. However, the micellar compactness also has some effect on the rotational motion of the dye. In the case of DTAB, ZnPcS₄ exhibits the highest fluorescence anisotropy value among the surfactants in the series, which could be because of greater penetration of the dye into DTAB micelles compared with the other two cationic surfactants.

Fig. 5 Plot of anisotropy values of ZnPcS₄ with increasing concentrations of TX-100 (a) and DTAB (b). Fluorescence emissions were monitored at the respective emission maxima. The inset shows the anisotropy values of ZnPcS₄ after the dissociation of larger aggregates.

Effect of micellar compactness on the rate of monomerization of ZnPcS₄

As discussed in earlier sections, in cationic micelles the percentage of ZnPcS₄ monomers is greater compared with that in an aqueous medium. The formation of monomers from larger aggregates of ZnPcS₄ in cationic micelles is greatly dependent on the hydrophobic chain length. As the monomeric form of ZnPcS₄ is solely responsible for its fluorescence, an increase in the concentration of the monomer will be reflected in an increase in the emission intensity. In DTAB micelles, ZnPcS₄ displays higher fluorescence intensity than in the other cationic micelles (TTAB and CTAB) (Fig. 6). This observation is also reflected in fluorescence lifetime data (Table 2), where the percentage of monomers follows the order of DTAB (43.63%) > TTAB (13.31%) > CTAB (8.48%). The change in the percentage of monomers of the fluorophore in cationic micelles with different chain lengths may be ascribed to changes in the microenvironment around the probe, which in some way force it to disaggregate into monomeric form. The effect of varying the chain length of a surfactant results in changes in its aggregation number, compactness of micelles and micellar hydration. It is a known fact that water can penetrate into a micelle to a certain extent depending upon the compactness of the micelle.^36–39 Micelles with longer chain lengths are more compact compared with those with shorter chains and hence experience less water penetration. The increase in the monomer concentration with a reduction in chain length cannot be simply explained by micellar hydration but rather by the degree of penetration of the probe into less compact micelles (DTAB). Fluorescence lifetime data also support this observation, as, with a decrease in compactness, the excited-state lifetime of the dye increases and follows the order of 3.33 ns > 3.19 ns > 2.93 ns for DTAB, TTAB and CTAB, respectively. The longest lifetime of the probe is found in DTAB micelles compared with the other cationic micelles, which is probably because of low micropolarity around the dye in DTAB micelles owing to high penetration inside the micelle. As ZnPcS₄ is a completely planar molecule, it may possess a low dipole moment. This low dipole moment helps to stabilize ZnPcS₄ in a less polar region and hence with a decrease in the compactness of a micelle it tends to penetrate more inside the micelle, resulting in monomerization of the dye.

Fig. 6 Plot of variation in the fluorescence intensity of ZnPcS₄ with increasing concentrations of CTAB, TTAB, and DTAB at the respective emission maxima.

Interaction of aziridinyl quinone (AZDCIQ) with ZnPcS₄ in an aqueous medium as well as different micellar microenvironments

Phthalocyanines are PDT-active dyes, which are frequently used as photosensitizers where molecular oxygen is essential to kill the cell (type II pathway). Tumour cells are hypoxic in nature, so the direct killing of tumour cells via the type II mechanism is limited. To overcome this limitation, Alegria et al. proposed photoreducible DNA alkylating quinones, which have a redox potential nearly the same as or more positive than that of oxygen. According to this hypothesis, the macrocyclic dye activates the quinone by photoreduction, which ultimately results in DNA alkylation. The success of the above-mentioned hypothesis depends on the extent and nature of the interaction between the dye and the aziridinyl quinone (AZDCIQ). In this scenario, the interaction of PDT-active ZnPcS₄ with the DNA alkylating agent AZDCIQ is studied in a deaerated aqueous medium, as well as neutral and different cationic micellar microenvironments, using steady-state absorption and emission studies. Because ZnPcS₄ failed to display any type of interaction with anionic SDS, this system was not considered for dye–quinone interaction studies.

The absorption and emission spectra of ZnPcS₄ were recorded with the gradual addition of AZDCIQ in a deaerated aqueous medium and different micellar microenvironments. As the AZDCIQ concentration increased, the emission intensity of ZnPcS₄ significantly decreased in all systems irrespective of the ionic character of the surfactant. This quenching of fluorescence can be accounted for in terms of the strong interaction between the fluorophore and the quinone molecule (Fig. 7). The extent of the interaction between AZDCIQ and PDT-active ZnPcS₄ has been estimated by calculating the association constant of AZDCIQ in the above-mentioned systems using the following equation:

where F₀ and F are the fluorescence emission intensities in the absence and presence of AZDCIQ, [Q] is the concentration of AZDCIQ, and K_a is the association constant. The values of the association constant of ZnPcS₄ with AZDCIQ in aqueous, TX-100, DTAB, TTAB, and CTAB media were found to be 5.13 × 10⁵ M⁻¹, 6.55 × 10⁴ M⁻¹, 5.88 × 10⁵ M⁻¹, 5.56 × 10⁷ M⁻¹, and 2.27 × 10⁶ M⁻¹, respectively (Fig. 8 and ESI†). The dye–quinone association constant was found to be greater in the cationic micelles and hence the interaction of ZnPcS₄ with AZDCIQ was replicated in hypoxic conditions and at the pH of tumor cells (5.6) in the series of cationic micelles to mimic the tumor cell environment. However, similar results in terms of variations in the fluorescence spectra as well as the association constants (K_a for H₂O, TTAB, CTAB and DTAB was 4.48 × 10⁵ M⁻¹, 2.14 × 10⁷ M⁻¹, 5.56 × 10⁶ M⁻¹, and 2.85 × 10⁵ M⁻¹, respectively) were obtained at pH values of 5.6 (Fig. S5 and S6†) and 7.4, which indicates that the interaction of the alkylating quinone AZDCIQ with the PDT-active dye ZnPcS₄ may remain equally strong in a tumor-like environment.

Fig. 7 Emission spectra of ZnPcS₄ (1.89 μM) (λ_ex at 344 nm) with increasing AZDCIQ concentrations in deaerated TTAB (15.53 mM) at 298 K.

Fig. 8 Plot of log(F₀ − F/F) versus log[AZDCIQ] for the determination of the association constant (K_a) of ZnPcS₄ with AZDCIQ in water and TTAB.

The differences in the values of K_a of the alkylating quinone with ZnPcS₄ in the aqueous and different micellar media are due to dissimilarities in the accessibility of quinone to the probe molecule in a confined medium. The highest association constants are observed in cases of cationic micellar media compared with an aqueous medium, as well as a neutral TX-100 medium. This is probably because the quinone molecules may reside near to the Stern layer of a cationic micelle, where the chance of interaction with ZnPcS₄ (which is also located in the Stern layer in a cationic micelle) is greater owing to entrapment in the surrounding environment. The fact that the value of K_a is relatively lower in the case of a TX-100 micelle is probably because of less availability of the dye (as it mostly resides in the apolar palisade or deeper core regions). It is pertinent to mention that the association constants in different cationic micelles do not follow any particular trend. The highest value of K_a is found in a TTAB micellar medium, which is because AZDCIQ is present in exactly that region where the dye resides in a TTAB micelle. In the case of DTAB, the dye penetrates a little deeper inside a micelle (because of less compactness in a DTAB micellar system), which also decreases the probability of interaction. Hence, the association constant of the alkylating quinone with the dye is found to be less. The value of K_a in any cationic micellar medium is much higher than in an aqueous medium, which consequently satisfies the ultimate aim of this experiment.

In order to gather more information on the excited-state dynamics, i.e., the nature of the interaction that exists between ZnPcS₄ and AZDCIQ molecules, TRF experiments were also performed. In these measurements, it was noted that on the gradual addition of AZDCIQ to aqueous and TX-100 media, as well as the three cationic micellar systems, the nature of the fluorescence decay and the excited-state lifetime of ZnPcS₄ remained unchanged throughout the experiment. Thus, the TRF experiment indicates that only static types of interaction occur between these two interacting partners.

Dynamic light scattering measurements

The effects of the negatively charged dye on the hydrodynamic diameter of different micellar systems were determined by DLS studies. The variations in the hydrodynamic diameter (d_h) of different micelles in the absence and presence of the anionic dye are illustrated in Fig. 9. From the DLS data (Table 3) for different micelles, it is observed that the average diameter of all the cationic micelles of varying chain lengths lies in the range of 2.5–3 nm; however, the average value of d_h for TX-100 (8.72 nm) is slightly greater than that of the cationic micelles under study. These results are in line with the reported literature.^40–42

Fig. 9 Dynamic light scattering graphs of TX-100, CTAB, TTAB, and DTAB in the absence and presence of ZnPcS₄: [TX-100] = 3.0 mM; [CTAB] = 3.65 mM; [TTAB] = 12.73 mM; [DATB] = 26.53 mM.

Table 3 Hydrodynamic diameters of different micelle systems at room temperature

Sr. no.	Micelle	dh/nm	Micelle–dye	dh/nm	Micelle–dye–quinone	dh/nm
1	CTAB	2.69	CTAB–ZnPcS4	295.0	CTAB–ZnPcS4–AZDCIQ	255.0
2	TTAB	2.69	TTAB–ZnPcS4	255.0	TTAB–ZnPcS4–AZDCIQ	220.0
3	DTAB	3.12	DTAB–ZnPcS4	21.04	DTAB–ZnPcS4–AZDCIQ	13.54
4	TX-100	8.72	TX-100–ZnPcS4	8.72	TX-100–ZnPcS4–AZDCIQ	10.10

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The addition of anionic ZnPcS₄ to neutral TX-100 does not bring about any noticeable change in the value of d_h but for the series of cationic micelles the value of d_h dramatically increases and follows the order of CTAB–ZnPcS₄ > TTAB–ZnPcS₄ > DTAB–ZnPcS₄. The observed variations in the value of d_h might be due to a transition from micelles to micellar clusters (aggregated micelles). The formation of micellar clusters is perhaps induced by the negatively charged macrocycle in its stacked dimer form in a fully micellised system (which may be justified by the excited-state lifetime (τ₂) being slightly longer in micelles compared with the bulk aqueous solution²⁸), which attracts a positively charged micelle (either dye-free or containing monomeric ZnPcS₄) towards itself.

This trend in the size distribution profile of cationic micelle clusters obtains its justification from Coulomb's law and is closely correlated with the location of the dye, which varies with the compactness of the micelles. As discussed in an earlier section, when the micellar compactness decreases it becomes easier for macrocyclic ZnPcS₄ to penetrate deeper inside the micelle. Therefore, it is observed that with the less compact DTAB negatively charged ZnPcS₄ is buried inside the palisade layer, which consequently weakens the electrostatic attraction toward other DTAB micelles and thereby minimizes the formation of clusters. However, with the more compact CTAB, where ZnPcS₄ is located in the Stern layer, the formation of micellar clusters is induced by the distance between the two interacting partners being relatively shorter. The trend in the formation of clusters in different cationic micelles is also in line with the percentage of ZnPcS₄ dimers present in the system. The presence of anionic dimers possibly increases the net negative charge over the dye–micelle system, which essentially assists the micellised dimer to attract another cationic micelle towards itself and form a micellar cluster easily.

Furthermore, the addition of AZDCIQ to dye–micelle systems gave rise to a slight decrease in the hydrodynamic diameter of all the micellar systems under study (Fig. S7,† Table 3). This is probably because of the competition that existed between the alkylating quinone and the cationic micelles towards the anionic macrocycle. However, in the case of TX-100 there was no change in the value of d_h with the addition of AZDCIQ, as there was no formation of clusters initially owing to the localisation of the dye in the deeper palisade layer of the non-ionic micelle.

Effect of central metal ion on the photophysical properties of phthalocyanine

Sulphonated metallophthalocyanines are readily aggregated in water owing to the π–π stacking interaction between the hydrophobic macrocyclic rings in two or more molecules. The introduction of a central metal ion into a phthalocyanine not only influences its photophysical properties but may also have a pronounced effect on the aggregation behaviour of the phthalocyanine.^9,31 Hence, a detailed comparison has been made between two phthalocyanines bearing two different central atoms (Al and Zn) in aqueous and different micellar environments. The interaction of AlPcS₄ with different micelles and an alkylating quinone has already been reported by our group.⁹ As discussed earlier, in water ZnPcS₄ predominantly remains in dimeric form. This could be because ZnPcS₄ has a perfect planar geometry, which easily allows other molecules to form π–π stacking interactions and hence favours aggregation. However, this aggregation behaviour is not observed in the case of AlPcS₄, where the dye remains solely in monomeric form in an aqueous medium. In AlPcS₄, the central aluminium atom coordinates axially to a chloride ion, resulting in a slightly distorted structure, which prevents any stacking interaction between two AlPcS₄ monomer molecules. Moreover, owing to this slightly distorted geometry, the dipole moment of AlPcS₄ is expected to be greater than that of planar ZnPcS₄. The lower dipole moment of ZnPcS₄ makes it slightly more hydrophobic in nature than AlPcS₄; hence, on increasing the concentration of TX-100 in an aqueous solution of ZnPcS₄, the dye starts to disaggregate owing to hydrophobic interaction between the surfactant and the ZnPcS₄ molecules. However, the addition of TX-100 to an AlPcS₄ solution does not produce any appreciable changes in its absorption or emission spectra, which suggests that AlPcS₄ is in monomeric form only. This phenomenon obtained further confirmation from TRF measurements. The aggregation–disaggregation patterns of both dyes showed a similar trend with cationic surfactants. The effect of cationic surfactants on the emission intensity of AlPcS₄, as well as that of ZnPcS₄, shows a similar trend, namely, that with a decrease in the surfactant chain length the emission intensity increases. However, in the case of AlPcS₄, the percentage of monomers was 100% in a fully micellized system with all cationic surfactants (CTAB, DTAB, and TTAB); hence, the increase in emission intensity is because of the alteration in the microenvironment (micellar hydration) around the probe.⁹ On the other hand, the percentage of ZnPcS₄ monomers increases with the decreasing compactness (or increasing micellar hydration) of micelles, which is responsible for the remarkable increase in fluorescence intensity. The increase in the percentage of monomers is because of greater penetration of ZnPcS₄ into micelles owing to its hydrophobic nature, which results in the dissociation of dimers into monomers, whereas AlPcS₄ prefers to reside near the Stern layer only, where micellar hydration varies with changes in the hydrophobic chain length of the cationic surfactant. The value of the fluorescence anisotropy (r) of Al phthalocyanine in different cationic micellar media follows the trend CTAB > TTAB > DTAB, which is exactly the reverse of that of Zn phthalocyanine, in which case the value of r increases owing to greater penetration into less compact micelles (DTAB). As both dyes possess negative charge, it is natural to expect that the dyes prefer to stay near the cationic Stern layer owing to electrostatic attraction. However, as we provided an opportunity to both the dyes to move inside the cationic micelles by decreasing the micellar compactness, ZnPcS₄ moved more inside the hydrophobic region of the cationic micelles, whereas AlPcS₄ stayed near the Stern layer. This reflects the hydrophobic nature of ZnPcS₄, which prefers the palisade region to the hydrophilic Stern layer owing to strong hydrophobic attraction in less compact micelles. This is further confirmed by TRF measurements of the dyes in different cationic micelles, where the excited-state lifetime of monomeric ZnPcS₄ increases with a decrease in the compactness of the micelles, whereas in the case of AlPcS₄ the excited-state lifetime does not change significantly. Analysis of the interaction between AZDCIQ and these two macrocycles in an aqueous medium, as well as in different micellar media, is an interesting area to investigate. ZnPcS₄ has been found to be the best photosensitizer in terms of cytocidal activity compared with AlPcS₄.⁸ This is because the triplet quantum yield displayed by Zn phthalocyanine is higher than that of AlPcS₄ owing to the heavy atom effect, which ultimately results in a high singlet oxygen quantum yield.³¹ Interestingly, both dyes exhibit strong interactions with an alkylating quinone in cationic micelles compared with in an aqueous system. However, the association constant (K_a) thus obtained in an aqueous medium, as well as in different cationic micelles, was found to be greater in the case of ZnPcS₄ compared with AlPcS₄. Hence, the alkylation of DNA, which is the ultimate goal of this study, is also expected to be more extensive in the case of ZnPcS₄. Hence, as a concluding remark, ZnPcS₄ not only exhibits superiority over AlPcS₄ in terms of cell killing ability, but also exhibits a stronger association with DNA alkylating quinones than AlPcS₄ in biomimicking systems.

Materials and methods

Chemicals

The alkylating quinone 2,5-dichlorodiaziridinyl-1,4-benzoquinone (AZDCIQ), cetyltrimethylammonium bromide (CTAB), and Triton X-100 (TX-100) were obtained from Sigma-Aldrich Chemicals, USA. Zinc phthalocyanine tetrasulphonate (ZnPcS₄) was procured from Frontier Scientific, USA. Dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB) and sodium dodecylsulphate (SDS) were obtained from Spectrochem, India and Alfa Aesar, England, respectively. All the chemicals were used as received.

Instrumentation

Steady-state absorption and emission spectra were recorded at 298 K using a Jasco V-630 spectrophotometer equipped with Peltier accessories and a Jasco FP-8300 spectrofluorometer equipped with a temperature controller unit. All the measurements were carried out using a 1.0 cm quartz cell and an external slit width of 5.0 nm. Steady-state fluorescence anisotropy was also recorded using a Jasco FP-8300 spectrofluorometer with polarizer accessories. For the anisotropy measurements, the excitation and emission bandwidths were both set at 5.0 nm. The steady-state anisotropy was calculated by the following equation:

r = (I_VV − GI_VH)/(I_VV + 2GI_VH)

where I_VV and I_VH are the emission intensities obtained with the excitation polarizer oriented vertically and the emission polarizer oriented vertically and horizontally, respectively. The factor G is defined as I_HV/I_HH. Fluorescence lifetime measurements were performed with a PTI PicoMaster time-resolved spectrofluorometer (PTI, HORIBA, USA) using a light-emitting diode (340 nm) as a light source using the technique of time-correlated single-photon counting (TCSPC). The pulse width of the PTI light-emitting diode was typically in the range of 700–1100 ps (LED-dependent), whereas the PMD-2 detector had a response time of approx. 180 ps and the temporal resolution was about 813 fs, which was well designed to provide a uniform response over the entire area of the photocathode. The emission spectral range of the detector was 185–820 nm. The data were analysed using FelixGX 4.1.2 decay analysis software provided by PTI (HORIBA, USA). The exponential fittings were authenticated by the reduced χ² criterion and the randomness of the fitted function to the raw data. The average fluorescence lifetime 〈τ〉 for biexponential decay of the dye was calculated using decay times (τ_i) and the relative contributions of components (α_i) by the following equation:⁴³

DLS measurements were performed at a scattering angle of 173° using a Malvern Nano-ZS 90 instrument (model DLS-Nano ZS, Zetasizer Nano Series) with a 4 mW He–Ne laser (λ = 632.8 nm). All measurements were taken at 298 K. Samples were filtered thoroughly through a 0.2 μm Millipore membrane filter prior to measurements to avoid the possible presence of any dust particles. The hydrodynamic diameters of the micelles and micelle–ZnPcS₄ systems were predicted from the intensity autocorrelation function of the time-dependent fluctuation in the intensity of scattered light from a suspension of particles undergoing random Brownian motion. The hydrodynamic diameter is determined by using the following equation:

d_h = k_BT/3πηD

where d_h is the hydrodynamic diameter, k_B is the Boltzmann constant, η is the solvent viscosity, and D is the translational diffusion coefficient.

All of the above experiments were repeated in order to obtain reproducible results.

Sample preparation

Millipore water was used for preparing all the solutions and the pH was adjusted as required (5.6 and 7.4). Oxygen was excluded from the samples by maintaining a positive pressure of nitrogen during all the experiments. Deoxygenation was stopped only while recording the absorption and emission spectra. A stock solution of ZnPcS₄ was prepared in Millipore water and a stock solution of AZDCIQ was prepared by adding it to Millipore water followed by warming, cooling and filtration. The different surfactant solutions were prepared by dissolving the required amounts of DTAB, TTAB, or CTAB in Millipore water at pH values of 7.4 and 5.6.

Conclusion

In summary, the present work exemplifies the systematic study of the aggregation pattern of ZnPcS₄ and its interaction with a DNA alkylating quinone in different biomimicking systems. We also highlighted the effect of the central metal ion in the above-mentioned studies by comparing the two most important water-soluble photosensitizers, i.e., ZnPcS₄ and AlPcS₄. In contrast to AlPcS₄, planar ZnPcS₄ is mostly present in dimeric form in aqueous solution, which ultimately reduces its PDT activity. In cationic micelles both dyes form larger aggregates in the premicellar concentration region. These larger aggregates of AlPcS₄ completely disaggregate into monomeric form in fully micellized systems, whereas in the case of ZnPcS₄ larger aggregates dissociate into both monomeric and dimeric forms. For ZnPcS₄, the percentage of monomers increases on a decrease in the compactness of cationic micelles, owing to progressively greater penetration into the less polar regions of the micelles. On the gradual addition of a neutral surfactant, the dimeric form of ZnPcS₄ progressively dissociates into monomers, with a significant enhancement in the monomeric emission band, to create two populations of monomeric dye molecules, one at the shear surface and the other in the deeper hydrophobic core of non-ionic TX-100 micelles. The anionic dye localizes itself in the Stern layer of cationic micelles; however, in contrast to AlPcS₄, ZnPcS₄ penetrates deeper inside cationic micelles on a decrease in the compactness of the micelles. On the addition of anionic ZnPcS₄, cationic micelles form micellar clusters owing to electrostatic interaction and the size of the micellar clusters varies linearly with the surfactant chain length. The interaction of ZnPcS₄ with a DNA alkylating quinone is found to be more prominent in a cationic micellar medium owing to the greater accessibility of the quinone to the probe molecule in a confined micellar microenvironment. The association constant of ZnPcS₄ with AZDCIQ is found to be greater than that of AlPcS₄ in aqueous as well as in micellar microenvironments. The increased dye–quinone interaction in confined micellar media may enhance the probable photosensitized reduction of quinone molecules and their subsequent DNA alkylation process. This consolidated spectroscopic research delivers an impressive future prospect of PDT by the use of a metallophthalocyanine and a DNA alkylating quinone together, especially in the hypoxic environment of a solid tumour.

Acknowledgements

SKG gratefully acknowledges the financial support from the Department of Science and Technology, India (Project no. SR/FT/LS/-172/2009). MJ gives thanks to VNIT for providing the research fellowship. The authors thank Prof. S. C. Bhattacharya, Jadavpur University, Kolkata, for providing the dynamic light scattering facility.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra13151d

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