Photosensitization Ability of a Water Soluble Zinc(II) - ACS Publications

J. Phys. Chem. B 2008, 112, 15701–15707

15701

Photosensitization Ability of a Water Soluble Zinc(II)tetramethyltetrapyridinoporphyrazinium Salt in Aqueous Solution and Biomimetic Reverse Micelles Medium Tomas C. Tempesti,† Juan C. Stockert,‡,§ and Edgardo N. Durantini*,† Departamento de Quı´mica, UniVersidad Nacional de Rı´o Cuarto, Rı´o Cuarto, Agencia Postal Nro. 3, X5804BYA Rı´o Cuarto, Co´rdoba, Argentina, Departamento de Biologı´a, Facultad de Ciencias, UniVersidad Auto´noma de Madrid, c/Darwin 2, Citologı´a A-114, Cantoblanco, E-28049 Madrid, Spain, and Centro de InVestigaciones Biolo´gicas, CSIC, E-28040 Madrid, Spain ReceiVed: September 11, 2008; ReVised Manuscript ReceiVed: October 15, 2008

The spectroscopic properties and the photodynamic activity of a highly water soluble zinc(II)tetramethyltetrapyridino[2,3-b:2′,3′-g:2′′,3′′-l:2′′′,3′′′-q]porphyrazinium salt (ZnTM2,3PyPz) were investigated in aqueous homogeneous solution and in biomimetic reverse micelles medium bearing photooxidizable biological substrates. Absorption and fluorescence spectroscopic studies indicate that ZnTM2,3PyPz is dissolved as monomer in water and in n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT, 0.1 M)/water (W0 ) 30) micellar system. Fluorescence quantum yields (ΦF) of 0.29 and 0.27 were calculated for ZnTM2,3PyPz in water and in AOT micelles, respectively. Spectroscopic analysis at different AOT concentrations showed interaction between ZnTM2,3PyPz and AOT reverse micelles with a binding constant (Kb) of 1.7 × 103 M-1. The photosensitization ability of ZnTM2,3PyPz was evaluated using 9,10-dimethylanthracene (DMA). Singlet molecular oxygen, O2(1∆g), production yielded values of Φ∆ ) 0.65 for ZnTM2,3PyPz in AOT micelles. Also, ZnTM2,3PyPz induced efficiently the decomposition of the amino acid L-tryptophan (Trp) and the nucleotide guanosine 5′-monophosphate (GMP) in both media. A value of ∼3.6 × 107 s-1 M-1 was found for the second order rate constant of Trp (krTrp) decomposition in the AOT system, which is near to that found in pure water. Moreover, ZnTM2,3PyPz formed stable complexes with GMP with a binding constant of KGMP ) 1.0 × 103 M-1. 1H NMR studies indicated that ZnTM2,3PyPz interacts mainly with the guanine moiety more than the sugar part of GMP. On the other hand, the photodynamic activity of ZnTM2,3PyPz produced decomposition of GMP. Quantification of GMP by HPLC indicates that the nucleotide is GMP photooxidized with a kobs ) 2.6 × 10-4 s-1 in water. Photooxidation of GMP considerably increases in deuteriated water indicating that ZnTM2,3PyPz appears to perform its photosensitizing action via the intermediacy of O2(1∆g). Also, efficient sensitized decomposition was observed in micellar media resembling that in pure water. These results provide a better understanding of the effective photodynamic action produced by ZnTM2,3PyPz like a potential phototherapeutic agent for the treatment of neoplastic diseases by photodynamic therapy. Introduction Phthalocyanine derivatives have a potential use as phototherapeutic agents in photodynamic therapy (PDT) of cancer and other medical applications.1-4 These photosensitizers exhibit a high absorption coefficient in the visible region of the spectrum, mainly in the phototherapeutic window (600-800 nm) and a long lifetime of triplet excited-state to produce efficiently singlet molecular oxygen, O2(1∆g).5 In general, PDT approach utilizes a combination of light, a photosensitizer and oxygen to achieve a cytotoxic effect that induces cell death. In photodynamic process, the photosensitizer excited-state can react directly with biomolecules to produce free radicals (type I reaction) or with molecular oxygen to produce highly reactive O2(1∆g) (type II reaction).6,7 Solubilization of photosensitizers in water plays an important role in biological processes. One of the main problems that affect * Corresponding author. Phone: +54 358 4676157. Fax: +54 358 4676233. E-mail address: [email protected]. † Universidad Nacional de Rı´o Cuarto. ‡ Universidad Auto´noma de Madrid. § Centro de Investigaciones Biolo´gicas.

the sensitizing ability of the phthalocyanines is the aggregation tendency due to the large π conjugate systems.5 Aggregation reduces the lifetimes of sensitizer excited state, probably due to enhanced radiationless excited-state dissipation, and therefore lowers the quantum yield of the triplet excited-state and of O2(1∆g) generation. Thus, the formation of aggregations precludes the photodynamic activity. The high versatility of phthalocyanines allows the substitution of some of their four-isoindole units by other nitrogenated heterocycle moieties, giving rise to different macrocycles azaanalogues.8 In the case of pyridine derivatives, these compounds can be quaternized, introducing substituents in order to modulate the solubility of the tetracationic derivatives.9-12 This procedure permits to form water soluble compounds, some of them solubilized as monomers in aqueous solutions, whereas others mainly remain as aggregates in several media. In previous studies, we have investigated the spectroscopic and photodynamic properties of zinc(II)tetramethyltetrapyridino[3,4-b:3′,4′-g:3′′,4′′-l:3′′′,4′′′-q]porphyrazinium tetraiodide (Scheme 1, ZnTM3,4PyPz) in diverse media.12,13 This phthalocyanine is aggregated in several solvents, including micellar

10.1021/jp808094q CCC: $40.75  2008 American Chemical Society Published on Web 11/17/2008

15702 J. Phys. Chem. B, Vol. 112, No. 49, 2008 SCHEME 1: Molecular Structures of ZnTM2,3PyPz and ZnTM3,4PyPz

systems with low water content. However, partial monomerization takes place increasing the amount of water dispersed in n-heptane/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/water micellar system, but extensive aggregation was found in pure water. ZnTM3,4PyPz was dissolved as a mixture of at least two species in methanol, aqueous sodium dodecylsulfate (SDS), and in methanol-pyridine mixtures.11 Therefore, this cationic porphyrazine is not an efficient photosensitizer in pure water.11,13 In the present work, we explore the spectroscopic properties and the photo-oxidation of biological substrates catalyzed by zinc(II)tetramethyltetrapyridino[2,3-b:2′,3′-g:2′′,3′′-l:2′′′,3′′′q]porphyrazinium (ZnTM2,3PyPz, Scheme 1) in water and in n-heptane/AOT/water micellar system. The main difference between ZnTM2,3PyPz and ZnTM3,4PyPz sensitizers is found in their solubilities as monomeric species in aqueous solutions. The studies were also extended to reverse micelles, which can be used as an interesting model to mimic the water pockets often found in various bioaggregates.14-16 In these media, the photodynamic activity of ZnTM2,3PyPz was analyzed in the presence of L-tryptophan (Trp) or guanosine 5′-monophosphate (GMP). Proteins are one of the cellular constituents that are susceptible to photooxidation reactions. In particular, the amino acid Trp is an important target in the cell, which is highly susceptible to many oxidizing agents.17-19 Modification of amino acid side chains can cause significant alterations in the physicochemical properties of proteins and can subsequently lead to cellular damage. Moreover, guanine is believed to be the site of O2(1∆g) attack in the photodynamic decomposition of DNA.20-24 It was found that complexes of guanine with efficient cationic photosensitizers produce photosensitized cleavage of DNA.25-27 The photodamage can be initiated by electron and/ or energy transfer between the excited sensitizer to a near base pair or by O2(1∆g) formed in close proximity to DNA.28 Therefore, this investigation provides further understanding about the characteristic of ZnTM2,3PyPz as photosensitizer with possible application in PDT. Materials and Methods General. UV-visible and fluorescence spectra were recorded on a Shimadzu UV-2401PC spectrometer and on a Spex FluoroMax fluorometer, respectively. Proton nuclear magnetic resonance spectra were recorded on a FT-NMR Bruker Advance 200 spectrometer at 200 MHz. The HPLC experiments were performed in a Waters 1525 liquid chromatograph equipped with a Varian 2550 UV-visible variable-wavelength detector and Phenomenex Luna C18 (5 µm, 150 × 4.60 mm) column. The light fluence rate was determined using a Radiometer Laser Mate-Q, Coherent, Santa Clara, CA. The refractive indexes (η) were measured using an Atago NAR-1T (Tokyo, Japan) refractometer. All the chemicals from Aldrich (Milwaukee, WI) were used without further purification. Sodium bis(2-ethylhexyl)-

Tempesti et al. sulfosuccinate (AOT) from Sigma (St. Louis, MO) was used as received. Solvents (GR grade) from Merck were distilled. Ultrapure water was obtained from Labconco (Kansas, MO) equipment model 90901-01. Photosensitizers. Zinc(II)phthalocyanine (ZnPc) and 5,10, 15,20-tetra(4-N,N,N-trimethylammoniumphenyl)porphyrin ptosylate (TMAP) were purchased from Aldrich. According with the synthetic procedure, phthalocyanine macrocycles were obtained as a mixture of the corresponding regioisomers. Zinc(II)tetramethyltetrapyridino[2,3-b:2′,3′-g:2′′,3′′-l:2′′′,3′′′q]porphyrazinium methylsulfate (ZnTM2,3PyPz) and zinc(II) tetramethyltetrapyridino[3,4-b:3′,4′-g:3′′,4′′-l:3′′′,4′′′-q]porphyrazinium tetraiodide (ZnTM3,4PyPz) were synthesized as previously described.9,12 Spectroscopic Studies. Spectra were recorded using 1 cm path length quartz cells at (25.0 ( 0.5) °C. The fluorescence quantum yields (ΦF) of phthalocyanines were calculated by comparison of the area below the corrected emission spectrum with that of TMAP as a fluorescence reference, exciting at λexc)590 nm.29 A value of ΦF ) (0.12 ( 0.01) for TMAP in n-heptane/AOT(0.1 M)/water (W0 ) 30) was calculated by the comparison with the fluorescence spectrum in water using ΦF ) 0.12 and taking into account the refractive index of the solvents, η(water) ) 1.333; η(n-heptane/AOT(0.1 M)/water (W0 ) 30)) ) 1.389.30 Studies in AOT Reverse Micelles. Studies in reverse micelles were performed using a stock solution of AOT (0.1 M), which was prepared by weighing and dilution in n-heptane. The addition of water to the corresponding solution was performed using a calibrated microsyringe. The amount of water present in the system was expressed as the molar ratio between water and the AOT present in the reverse micelle (W0 ) [H2O]/ [AOT]). In all experiments, W0 ) 30 was used. The mixtures were sonicated for 10-15 s to obtain perfectly clear micellar system. The binding constants, Kb ) [phthalocyanineb]/[phthalocyaninef][AOT] (where the terms [phthalocyanineb] and [phthalocyaninef] refer to the concentration of bound and free phthalocyanine, respectively, and [AOT] is the total surfactant concentration) were calculated from the spectral changes at Q-band varying AOT concentration using Ketelaar’s equation (eq 1):14

1 1 ) + A - AHp (εb - εHp)[phthalocyanine]0 1 (1) (εb - εHp)[phthalocyanine]0Kb[AOT] where [phthalocyanine]0 is the total concentration of the phthalocyanine, A is the absorbance at different [AOT], AHp is the absorbance in n-heptane, εb and εHp are the molar absorptivity for the phthalocyanine bound to the interface and in the organic medium, respectively. Plotting the left-hand side term of eq 1 vs 1/[AOT], the value of Kb is obtained from the intercept and the slope ratio. Equation 1 was also used with fluorescence emission date. Phthalocyanine Binding to GMP. Stock solution of GMP (10 mM) was prepared by weighing and dilution in water. Absorbance titrations were conduced by adding concentrated stock solution of GMP directly to a cuvette containing phthalocyanine solution (2 mL, ∼2 µM) in water. The apparent binding constants (KGMP) for phthalocyanine-GMP complex were calculated from the absorbance changes at the Q maximum

Photosensitization Ability of ZnTM2,3PyPz

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(∆A) assuming a 1:1 stoichiometry and that the nucleotide concentration is always significantly larger than the phthalocyanine concentration using eq 2, where (∆A)∞ represents the extrapolated absorbance change at [GMP] f ∞.31,32

1 1 1 + ) ∆A (∆A)∞ (∆A)∞KGMP[GMP]

(2)

A plot of 1/∆A vs 1/[GMP] was used to calculate the value of KGMP from the ratio of the intercept to the slope. The binding of ZnTM2,3PyPz to GMP was followed by 1H NMR. NMR experiments were performed using 5 mm NMR tube with 3 mM GMP in 1 mL D2O. Different concentration (0-30 µM) of ZnTM2,3PyPz were added from a stock solution in D2O. Steady State Photolysis. Solutions of 9,10-dimethylanthracene (DMA, 35 µM) or L-tryptophan (Trp, 25 µM) and photosensitizer in different media were irradiated in 1 cm path length quartz cells (2 mL) with monochromatic light at λirr ) 640 nm for ZnTM2,3PyPz and λirr ) 667 nm for ZnPc (sensitizer absorbance 0.2), from a 75 W high-pressure Xe lamp through a high intensity grating monochromator, Photon Technology Instrument. The light fluence rate was determined as 1.5 mW/cm2. The kinetics of DMA and Trp photooxidation were studied by following the decrease of the absorbance (A) at λmax ) 378 nm and the fluorescence intensity (F) at λ ) 350 nm, respectively. The observed rate constants (kobs) were obtained by a linear least-squares fit of the semilogarithmic plot of ln A0/A or ln F0/F vs. time. Photooxidation of DMA was used to determine O2(1∆g) production by the photosensitizer.33 Measurements of the sample and reference under the same conditions afforded Φ∆ for phthalocyanines by direct comparison of the slopes in the linear region of the plots. ZnPc (Φ∆ ) 0.56) was used as references in DMF.34 The reaction rate constant of Trp photooxidation (krTrp) was calculated considering the mechanism described before for the photooxidation reactions in AOT reverse micelles,35,36 from eq 3, where K ) 0.11 and krDMA ) 0.8 × 107 s-1 M-1. DMA Trp DMA kTrp obs /kobs ) kr K/kr

Figure 1. Absorption spectra of ZnTM2,3PyPz in water (solid line) and in n-heptane/AOT (0.1 M)/water (W0 ) 30) (dashed line) and ZnTM3,4PyPz in n-heptane/AOT (0.1 M)/water (W0 ) 30) (dotted line).

TABLE 1: Spectroscopic Data for ZnTM2,3PyPz in Different Media properties Q λmax Q

(nm) ε (M-1 cm-1) Emission λmax (nm) ΦF a

water

micellesa

640 (9.1 ( 0.1) × 104 651 (0.29 ( 0.02)

638 (9.2 ( 0.1) × 104 653 (0.27 ( 0.02)

n-Heptane/AOT(0.1 M)/water (W0 ) 30).

(3)

Solutions of GMP (100 µM in water, 2 mL) and photosensitizer (A640 ) 0.2) were irradiated in quartz cuvettes with visible light from a Novamat 130 AF slide projector equipped with a 150 W lamp. The light was filtered through a 2.5 cm glass cuvette filled with water to absorb heat. A wavelength range between 350-800 nm was selected by optical filters. The light fluence rate at the treatment site was 30 mW/cm2. The disappearance of GMP was monitored by decrease in the absorption peak at 254 nm.31 Kinetic determination of GMP decomposition was determined by HPLC. The separation was performed using a mobile phase composed of 49% water/49% methanol/2% acetic acid (flow: 0.5 mL/min). Detection of GMP was achieved by UV-visible detector at 270 nm. All the experiment were performed at (25.0 ( 0.5) °C. The pooled standard deviation of the kinetic data, using different prepared samples, was less than 10%. Results and Discussion Absorption and Fluorescence Spectroscopic Studies. The absorption spectra of ZnTM2,3PyPz in water and n-heptane/ AOT(0.1 M)/water (W0 ) 30) are shown in Figure 1. The spectra show the typical Soret and Q-bands, characteristic of aza-

Figure 2. Fluorescence emission spectra of ZnTM2,3PyPz in water (solid line) and in n-heptane/AOT (0.1 M)/water (W0 ) 30) (dashed line) and ZnTM3,4PyPz in n-heptane/AOT (0.1 M)/water (W0 ) 30) (dotted line), λexc ) 590 nm.

analogues of zinc phthalocyanines.37 In AOT system, the spectrum shows two close maxima in the Q-band region, which indicate that the phthalocyanine is a statistical mixture of regioisomers produced by the synthetic method.9,11 The band absorption maxima and the values of molar coefficient (ε) of the ZnTM2,3PyPz are summarized in Table 1. For comparison, Figure 1 also shows the spectrum of ZnTM3,4PyPz in AOT reverse micelles. As can be observed, the Q-band of ZnTM2,3PyPz is blue shifts by ∼40 nm with respect to that of the ZnTM3,4PyPz analogue. The steady-state fluorescence emission spectrum of ZnTM2, 3PyPz was recorded in aqueous solution and in AOT micelles (Figure 2). The emission spectrum shows two bands in the red spectral region, which are characteristic for similar Zn(II) phthalocyanines (Table 1).12 Fluorescence quantum yields (ΦF)

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Figure 3. Fluorescence excitation spectra of ZnTM2,3PyPz in water (solid line) and in n-heptane/AOT (0.1 M)/water (W0 ) 30) (dashed line), λem ) 670 nm, and ZnTM3,4PyPz in n-heptane/AOT (0.1 M)/ water (W0 ) 30) (dotted line), λexc ) 710 nm.

of ZnTM2,3PyPz were calculated by comparison with a reference (Table 1). The values of ΦF are very similar in both media and agree with those of similar porphyrazine derivatives dissolved as monomers.11,12 As observed above by absorption, the emission band of ZnTM2,3PyPz is hypsochromically shifted by ∼40 nm with respect to that of ZnTM3,4PyPz (Figure 2). A small Stokes shift (∼10 nm) was observed indicating that the spectroscopic energy is nearly identical to the relaxed energy of the singlet state. Taking into account the porphyrazine energy of the 0-0 electronic transitions, the energy level of the singlet excited stated (Es) was calculated from the intersection of the normalized absorption and fluorescence spectra. A value of Es ) (1.92 ( 1) eV was found in both media and this result is expected because the Q-band is shifted to a higher energy than in ZnPc derivatives.38 The fluorescence excitation spectrum of the ZnTM2,3PyPz was measured in these media (Figure 3), monitoring the emission at 670 nm. Also, it shows the excitation spectra of ZnTM3,4PyPz in AOT system. Excitation spectrum shows sharp Q-bands and vibration satellites bands at 590-600 nm. This band structure is typical for phthalocyanine derivatives and the two peaks at 624 and 648 nm evidence the presence of a mixture of regioisomers. The spectrum of ZnTM2,3PyPz resemble the absorption spectra (Figure 1), indicating that these sensitizers are essentially unaggregated in this medium. Interaction with AOT Reverse Micelles. The solubilization and interaction of ZnTM2,3PyPz were spectroscopically analyzed in n-heptane/AOT/water (W0 ) 30) reverse micelles. When the absorption spectra of ZnTM2,3PyPz were studied at various AOT concentrations, an increase in the intensity of the Q-band was observed as the surfactant concentration increased (Figure 4A, inset). Comparable results were observed by fluorescence emission spectroscopy, increasing the surfactant concentration (Figure 4B, inset). This effect is attributed to the interaction between the cationic phthalocyanine and the AOT micelles. Plotting the left-hand side term of eq 1 vs 1/[AOT], the value of the binding constant is calculated from the ratio of slope and the intercept. Representative results for ZnTM2,3PyPz are shown in Figure 4. Using this procedure, values of Kb ) (1730 ( 200) and (1680 ( 200) M-1 were obtained by absorption and emission date, respectively. Both approaches produce the same values of Kb, considering the experimental error, and this result is indicative that ZnTM2,3PyPz is dissolved as monomer in the AOT micelles. The large value of Kb indicates that this tetracationic porphyra-

Figure 4. (A) Variation of 1/(A - AHp) as a function of 1/[AOT] for ZnTM2,3PyPz in n-heptane/AOT (0.1 M)/water (W0 ) 30) reverse micelles (λ ) 644 nm). (inset) Absorbance spectra for ZnTM2,3PyPz at different AOT concentrations. (B) Variation of 1/(I - IHp) as a function of 1/[AOT] for ZnTM2,3PyPz in n-heptane/AOT (0.1 M)/ water (W0 ) 30) reverse micelles (λ ) 654 nm). (inset) Fluorescence emission spectra for ZnTM2,3PyPz at different AOT concentrations. (dashed line) Linear regression fit by eq 1.

zine is strongly associated with the micellar interface. Since the spectroscopic properties of ZnTM2,3PyPz (Table 1) are very similar in water and in the AOT micellar system, and considering that at W0 ) 30 the water microenvironment resembles that of bulk water,39 it can be inferred that the sensitizer is mainly localized in the aqueous pool of the interface. Also, the Kb value of ZnTM2,3PyPz is considerably higher than (210 ( 10) M-1, which was found for ZnTM3,4PyPz under the same conditions.13 This behavior reflexes the higher affinity of ZnTM2,3PyPz by the aqueous polar interface of the AOT micelles. Photosensitized Decomposition of 9,10-Dimethylanthracene and O2(1∆g) Production. Photooxidation of DMA sensitized by ZnTM2,3PyPz was studied in n-heptane/AOT (0.1 M)/water (W0 ) 30) reverse micelles. Because DMA is a nonpolar compound, it is mainly solubilized in the organic phase (n-heptane) of the micellar system.40 In this microenvironment, the substrate reacts with the O2(1∆g) photogenerated by the cationic porphyrazine. From first-order kinetic plots of the DMA absorption at 378 nm with time (Figure 5A) the values of the observed rate constant (kobs) were calculated (Table 2). As can be seen, ZnTM2,3PyPz photodecompose DMA faster than ZnPc in the micelles. Taking into account that DMA quenches O2(1∆g) exclusively by chemical reaction, it is used as a method to

Photosensitization Ability of ZnTM2,3PyPz

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Figure 6. Variation of 1/(∆A) vs 1/[GMP] for spectral titration of ZnTM2,3PyPz with GMP in water, λmax ) 636 nm. (dashed line) Linear fit by eq 2. (inset) Absorption spectra of ZnTM2,3PyPz in water at different GMP concentrations (49, 99, 147, 196, 244, 291, 338, 385, and 430 µM). Values represent the mean ( standard deviation of three separate experiments.

Figure 5. (A) First-order plots for the photooxidation of (A) DMA (35 µM) photosensitized by ZnTM2,3PyPz (2) and ZnPc (•) in n-heptane/AOT (0.1 M)/water (W0 ) 30). (B) Trp (25 µM) photosensitized by ZnTM2,3PyPz in water (9) and in n-heptane/AOT (0.1 M)/ water (W0 ) 30) (2). Values represent the mean ( standard deviation of three separate experiments.

TABLE 2: Kinetic Parameters for Substrates Photodecomposition and Quantum Yield of O2(1∆g) Production (Φ∆) of ZnTM2,3PyPz in Different Media properties -1 a kDMA obs (s ) a Φ∆ -1 kTrp obs (s ) -1 kGMP (s ) obs

water

micellesa

(9.7 ( 0.1) × 10-5 (2.6 ( 0.1) × 10-4

(7.1 ( 0.1) × 10-5 0.65 ( 0.04b (3.6 ( 0.1) × 10-5 (1.8 ( 0.1) × 10-4

a n-Heptane/AOT(0.1 M)/water (W0 ) 30). -5 -1 reference kDMA s . obs ) (6.1 ( 0.1) × 10

b

Using ZnPc as

evaluate the ability of the sensitizers to produce O2(1∆g) in solution.41 Under this condition, O2(1∆g) quantitatively converts DMA into its corresponding endoperoxide, which can be used as detector of O2(1∆g) through its changes in the absorption spectrum.42 The quantum yield of O2(1∆g) production (Φ∆) was calculated by comparing the slope for the ZnTM2,3PyPz with that for the reference (ZnPc) from the plots shown in Figure 5A. The Φ∆ value for ZnTM2,3PyPz (Table 2) indicates that this phthalocyanine is an efficient photosensitizer to generate O2(1∆g) in the microheterogeneous media formed by reverse micelles of AOT. The photodynamic activity is higher than that previously found for ZnTM3,4PyPz (Φ∆ ) 0.5) under similar conditions.13 However, the values of Φ∆ can significantly change according to the medium, diminishing when the sensitizer is partially aggregated. Thus, there are limitations to predict

photodynamic activity of sensitizers in different media even though in the presence of substrates those have affinity for these sensitizers. Photooxidation of L-Tryptophan. The amino acid Trp was used as a substrate model for the compounds of biological interest that would be potential targets of phthalocyanine photodynamic action. This substrate can be efficiently photooxidized by both type I and type II reaction mechanisms.43,44 The photoprocess follows first-order kinetics with respect to Trp concentration, as showed in Figure 5B for [Trp] ) 25 µM. From Trp were calculated the plots in Figure 5B, the values of the kobs Trp for ZnTM2,3PyPz. The results in Table 2 indicate a lower kobs value in the AOT micelles in comparison with that found in water. In the AOT microheterogeneous system, Trp is exclusively solubilized in the polar side of the interface and it is oxidized by O2(1∆g).40,45 To evaluate the reaction rate constant of Trp photooxidation (krTrp) in this medium, DMA was used as actinometer in the same experimental conditions. From the ratio of the first-order slopes between the Trp and DMA using eq 3, the value of krTrp was calculated taking into account the mechanism described before for the photooxidation reactions in AOT reverse micelles.36 This procedure gives a value of krTrp ) (3.7 ( 0.1) × 107 s-1 M-1, which is higher than the values reported before krTrp ) 1.9 × 107 s-1 M-1 and krTrp ) 2.3 × 107 s-1 M-1 in a similar AOT reverse micellar system using porphyrins at W0 ) 10 (ref 40) and rose bengal at W0 ) 22.2 (ref 35), respectively. However, the present result for krTrp is even lower than the value reported in water (krTrp ) 6.1 × 107 s-1 M-1)35 indicating that Trp at this amount of water dispersion (W0 ) 30) is not able to quench O2(1∆g) in the water pool of AOT reverse micelles with the same effectiveness as in pure water. Interaction with GMP. Titration of ZnTM2,3PyPz with GMP in water produces the spectral changes in the Q-band, which are shown in Figure 6. The hypochromicity of Q-band observed for ZnTM2,3PyPz indicates that this cationic photosensitizer binds to GMP. Thus, the spectral analysis shows that ZnTM2,3PyPz has a high affinity for GMP. The apparent binding constant (KGMP) was calculated giving a value of (1.0 ( 0.1) × 103 M-1. This value is comparable with those previously reported for the interaction of tetracationic mesotetrakis(4-N-methylpyridyl)porphyrin (TMPyP) and 5,10,15,20-

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Figure 7. 1H NMR spectra of GMP in D2O at different ZnTM2,3PyPz concentrations (A) 0, (B) 5, (C) 10, (D) 15, (E) 20, (F) 25, and (G) 30 µM.

tetrakis(R-trimethylammonium-p-tolyl)porphyrin tetrabromide (KGMP ∼ 7 × 103 M-1).31 Also, a value of 1.4 × 104 M-1 was reported for the interaction of TMAP with GMP.27 These results suggest that tetracationic macrocycles, such as phthalocyanine or porphyrin derivatives, present an effective interaction with GMP. 1 H NMR spectroscopy was used to investigate the interaction of ZnTM2,3PyPz with GMP in aqueous solution. The 1H NMR spectrum of GMP in D2O contains two protons, H8 (8.04 ppm) and H1′ (6.19 ppm), which are useful in monitoring the interaction between the nucleotide and phthalocyanine. The remaining nonexchangeable sugar proton resonances occur between 3.8-4.6 ppm as overlapping multiplets and were not used to follow the interaction. Figure 7 shows the 1H NMR spectral changes of GMP in D2O at different concentration of ZnTM2,3PyPz. The signals for H8 were found to be shifted to downfield at 8.86 ppm, while H1′ was practically unchanged. This behavior shows that the ZnTM2,3PyPz interacts with GMP mainly through the guanine moiety. Inspection of the 1H NMR spectra confirmed that the sugar moiety was not modified upon interaction. Similar effect was previously found for the reaction between GMP and the platinum(II)-triamine complexes.46 Platinum(II) complexes binds to the N7 nitrogen of the guanine residue of GMP to form a coordinated bond with the Pt metal center. Photosensitized Decomposition of GMP. The nucleotide GMP was chosen because guanine is believed to be the site of 1 O2(1∆g) attack in the photodynamic damage to DNA.28,47 Photosensitization of GMP was compared in water and AOT micellar system. Continuous irradiation of ZnTM2,3PyPz with visible light in air-saturated condition leads to GMP decomposition as evidenced by the formation of a broad absorption band above 254 nm (Figure 8A, inset). It is not possible to calculate the decomposition rate constant of GMP from these absorption data because of the overlap of the spectra of GMP with its degradations products.48 However, photosensitized decomposition of GMP by absorption can be approximately estimated in water from decrease of the GMP peak at 254 nm as showed in Figure 8A. As can be seen, there is a clear tendency of GMP decomposition sensitized by ZnTM2,3PyPz. The time course of reactions was followed by HPLC using a reverse phase C18 column eluted with acidified methanol/water mixture. The peak at retention time 6 min was assigned to GMP via comparison with chromatograms of this compound alone. After different times of reaction, it was detected that the peak

Figure 8. Photosensitized decomposition of GMP (100 µM) sensitized by ZnTM2,3PyPz in water (A) from decrease of GMP absorption peak at 254 nm; (inset) difference absorption spectra of GMP after different irradiation times, intervals 10 min. (B) First-order plots for the photooxidation of GMP from HPLC analysis. Values represent the mean ( standard deviation of three separate experiments. GMP ) was of GMP decreases. The rate constant of reaction (kobs obtained from the integrated peak areas. The reaction was treated assuming pseudo first-order kinetics as shown in Figure 8B. The results in both media are summarized in Table 2. As can beobserved,thedecompositionofGMPsensitizedbyZnTM2,3PyPz is faster in water than in micelles, in similar way than that found with Trp. However, since cationic porphyrazine binds to GMP by electrostatic attraction, an electron transfer pathway may also be contributing to its decomposition under these conditions.49 In fact, type I photosensitization mechanism leads to oneelectron oxidation of nucleobase with a high preference for the guanine base that exhibits the lowest ionization potential among DNA components. This leads to degradation products such as 8-oxo-7,8-dihydroguanine (8-oxoGua) and related open imidazole ring compound, namely 2,6-diamino-4-hydroxy-5-formamidopyrimidine that are both issued from the same precursor.50 The type II mechanism which is usually the prevalent acting mechanism for phthalocyanine derivatives is operating through the generation of O2(1∆g). The only DNA component target for the reaction of O2(1∆g) is guanine and as a result 8-hydroperoxy2′-deoxyguanosine is formed in aqueous solutions, which is able to rearrange into spiroiminodihydantoin nucleosides. Also, 8-oxoGua is formed as a relatively minor product.24 To evaluate the O2(1∆g)-mediated photooxidation of GMP sensitized by ZnTM2,3PyPz, the reaction was performed in deuteriated water. This solvent was used to increase the lifetime

Photosensitization Ability of ZnTM2,3PyPz GMP of O2(1∆g).51 Under this condition, a value of kobs ) (45.3 ( -4 -1 0.1) × 10 s was obtained. Thus, the reaction rate is ∼17 times faster in D2O than in aqueous solution. This increase is very similar to that produced in the lifetime of O2(1∆g) in D2O respect to water.51 Therefore, these results evidence an important contribution of type II photosensitization in the GMP decomposition induced by this cationic sensitizer.

Conclusions Tetracationic ZnTM2,3PyPz exists essentially as monomer in aqueous systems, including water dispersion in AOT reverse micelles. This sensitizer intensely absorbs in the phototherapeutic window and emits fluorescence that allows its detection and quantification in cells. In AOT system, ZnTM2,3PyPz is mainly bound to the micellar interface and its spectroscopic properties resemble those in bulk water. This porphyrazinium salt presents a high Φ∆ and induced efficiently the decomposition of Trp and GMP in both media. Photodecomposition of the amino acid was faster in water; however, the reaction rate constant in the AOT system is near to that found in pure water. Moreover, ZnTM2,3PyPz formed stable complexes with GMP, interacting mainly with the guanine moiety of the GMP. Even though the photooxidation of GMP is effectively sensitized by this porphyrazine in water, the nucleotide decomposition increases considerably in deuteriated water, indicating that ZnTM2,3PyPz appears to perform its photosensitizing action via the intermediacy of O2(1∆g). Also, efficient sensitized decomposition was observed in the AOT microheterogeneous system. Therefore, these results demonstrate that the tetracationic ZnTM2,3PyPz offers a promising molecular architecture for photosensitizer agents with potential applications in cell inactivation by PDT. Acknowledgment. The authors thank Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET) and Agencia Nacional de Promocio´n Cientı´fica y Te´cnolo´gica (ANPCYT), Argentina, for financial support. E.N.D. is a Scientific Member of CONICET. T.C.T. thanks CONICET for a postdoctoral research fellowship. References and Notes (1) Castano, A. P.; Demidova, T. N.; Hamblin, M. R. Photodiagn. Photodynam. Therapy 2004, 1, 279–293. (2) Stockert, J. C.; Juarranz, A.; Villanueva, A.; Nonell, S.; Horobin, R. W.; Soltermann, A. T.; Durantini, E. N.; Rivarola, V.; Colombo, L. L.; Espada, J.; Can˜ete, M. Curr. Topics Pharmacol. 2004, 8, 185–217. (3) Durantini, E. N. Curr. BioactiVe Comp. 2006, 2, 127–142. (4) Jori, G. J. EnViron. Pathol. Toxicol. Oncol. 2006, 25, 505–519. (5) Nyokong, T. Coord. Chem. ReV. 2007, 251, 1707–1722. (6) Ochsner, M. J. Photochem. Photobiol. B: Biol. 1997, 39, 1–18. (7) DeRosa, M. C.; Crutchley, R. J. Coord. Chem. ReV. 2002, 233234, 351–371. (8) Stuzhin, P. A.; Ercolani, C. The Porphyrin Handbook, Phthalocyanines: Synthesis, Pophyrazines with Annulated Heterocycles; Kadish, K. M., Smith, K. M., Guilard, R., Eds.; Academic Press: San Diego, CA, 2003; Vol. 15, pp 263-364. (9) Wo¨hrle, D.; Gitzel, J. J. Chem. Soc. Perkin Trans 2 1985, 1171– 1177. (10) Seotsanyana-Mokhosi, I.; Kuznetsova, N.; Nyokong, T. J. Photochem. Photobiol. A: Chem. 2001, 140, 215–222. (11) Marti, C.; Nonell, S.; Nicolau, M.; Torres, T. Photochem. Photobiol. 2000, 71, 53–59. (12) Dupouy, E. A.; Lazzeri, D.; Durantini, E. N. Photochem. Photobiol. Sci. 2004, 3, 992–998.

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