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Chemical Modification of a Tetrapyrrole-Type Photosensitizer: Tuning Application and Photochemical Action beyond the Singlet Oxygen Channel Yasser M. Riyad, Sergej Naumov, Stanislaw Schastak, Jan Griebel, Axel Kahnt, Tilmann Häupl, Jochen Neuhaus, Bernd Abel, and Ralf Hermann J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 10 Sep 2014 Downloaded from http://pubs.acs.org on September 14, 2014

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The Journal of Physical Chemistry

Chemical Modification of a Tetrapyrrole-Type Photosensitizer: Tuning Application and Photochemical Action beyond the Singlet Oxygen Channel Yasser M. Riyad 1,2,*, Sergej Naumov3, Stanislaw Schastak5,6, Jan Griebel1, Axel Kahnt4, Tilmann Häupl1, Jochen Neuhaus7, Bernd Abel1,3, Ralf Hermann1,6

1

Wilhelm-Ostwald-Institute for Physical and Theoretical Chemistry, Faculty of Chemistry and

Mineralogy, University of Leipzig, Permoserstr. 15, 04318 Leipzig, Germany 2

Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, 11884, Cairo,

Egypt 3

Leibniz Institute of Surface Modification, Chemical Department, Permoserstr. 15, 04318

Leipzig, Germany 4

Friedrich-Alexander-Universität Erlangen-Nürnberg, Department of Chemistry and Pharmacy

and Interdisciplinary Center for Molecular Materials, Egerlandstr. 3, 91058 Erlangen, Germany 5

Department of Ophthalmology, Faculty of Medicine, Univeristy of Leipzig, Liebigstr. 10-14,

04103 Leipzig, Germany 6

Laser-Medical Center e.V., Liebigstr. 10-14, 04103 Leipzig, Germany

7

Department of Urology, University of Leipzig, Liebigstr. 20, 04103 Leipzig, Germany

1 Environment ACS Paragon Plus


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ABSTRACT: Reactive oxygen species (ROS) formed by light activated photosensitizers (PSs) are the hallmark of photodynamic therapy (PDT). It is generally accepted that commonly used PSs generate singlet oxygen (1O2) as the cell-toxic species via Type II photosensitization. We explored here the consequences of chemical modification and the influence of the net charge of a cationic tetrahydroporphyrin derivative (THPTS) relative to the basic molecular structure on the red-shift of absorption, solubility, mechanistic features, and photochemical as well as cell-toxic activity. In order to shed light into the interplay between chemical modification driven intra- and intermolecular photochemistry, intermolecular interaction, and function, a number of different spectroscopic techniques were employed and our experimental studies were accompanied by quantum chemical calculations. Here we show that for THPTS neither 1O2 nor other toxic ROS (superoxide and hydroxyl radicals) are produced directly in significant quantities in aqueous solution (although the formation of singlet oxygen is energetically feasible and as such observed in acetonitrile). Nevertheless, the chemically modified tetrapyrrole photosensitizer displays efficient cell toxicity after photoexcitation. The distribution and action of THPTS in rat bladder caricinoma AY27 cells measured with fluorescence lifetime imaging microscopy shows accumulation of the THPTS in lysosomes and efficient cell death after irradiation. We found evidence that THPTS in water works mainly via the Type I mechanism involving the reduction rather than oxidation of the excited triplet state THPTS(T1) via efficient electron donors in the biosystem environment and subsequent electron transfer to produce ROS indirectly. These intriguing structure-activity relationships may indeed open new strategies and avenues in developing PSs and PDT in general.


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KEYWORDS: Reactive oxygen species, bacteriochlorin, photochemistry, photodynamic therapy, in vivo studies, quantum chemistry.



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Introduction Photodynamic therapy (PDT) has been widely used with continuous success in therapeutic treatment of cancer over the past two decades for a number of malignant and non-malignant applications.1-7 The well established treatment relies on the combination of a photosensitizer (PS), visible or near infrared light, and molecular oxygen (O2), to generate cell-toxic short-living reactive oxygen species (ROS), such as oxygen containing radicals, e.g. superoxide (O2•―), hydroxyl radical (•OH) or reactive excited states of oxygen, i.e. singlet oxygen (1O2). The excited triplet state of a PS (PS(T1)) plays an important role in the generation of the ROS which can be formed in two different competing chemical reactions: a Type I and/or a Type II process.8 In a Type I pathway the PS(T1) reacts with the surrounding substrate via electron transfer, yielding radical ions or radicals such as O2•― or •OH respectively. Whereas a Type II process involves direct energy transfer from the PS(T1) to the ground state of oxygen to generate 1O2, where the triplet energy of PS(T1) must lie above 1O2 excitation energy (94.5 kJ mol-1).9, 10 Both processes can take place simultaneously and the relation between them depends on the PS, the substrate and the sensitizer’s environment. However, the primary generated ROS in both mechanisms initiating a cascade of events in the tumor tissue and cause cell death. It has been widely accepted that 1O2 formed via a Type II process is the major cytotoxic species responsible for the biological effect of PDT in the targeted cells.11-14 In this regard, potent PSs should have high yields of 1O2 photogeneration combined with high triplet quantum yield and long triplet lifetime. They should also possess strong absorption band within the phototherapeutic window (650-850 nm); where the tissue penetration depth of light is optimal, flanked by a high selectivity for malicious tissue. Extensive research based on molecular tuning of the chemical and electronic structure properties of PSs has been done, not only to tune their


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long-wavelength absorption maxima within the therapeutic window, but also to enhance the 1O2 yield.15-31 Now the question arises whether or not 1O2 photogeneration is necessarily prerequisite for a PS in PDT. Can we expect significant PDT efficiency by a PS, where its PS(T1) is energetically unable to generate 1O2? In this particular case the Type I reaction is the only alternative working rout which involves either photooxidation (reaction 1) or photoreduction (reaction 2a) of the PS(T1). In a photooxidation process a direct electron transfer may proceed from the PS(T1) to O2, whereas in a photoreduction reaction an electron transfer is induced from an endogenous electron donor (D) to the PS(T1). Both reaction channels result in the generation of the O2•― ions (reactions 1 and 2b) which then lead to subsequent formation of hydrogen peroxide (H2O2), •OH and other ROS.24,25,31-35 Also a radical cation (D•+, reaction 2a) may be the culprit for cell death via a peroxyl radical (DO2•, reaction 2d); caused by the reaction of oxygen with the product of reaction 2c; which then results in a subsequent chain or fragmentation reactions. Although conclusive proof for the occurrence of the Type I pathway is still limited, some recent reports have revealed that high concentrations of O2•―, •OH and other species formed via Type I mechanism can trigger highly toxic effects. However, high levels of these ROS can cause death of tumor cells by overwhelming their antioxidant defenses.36 To the best of our knowledge there is no systematic study that addresses the effect of energy level tuning via molecular structure modification of any particular PS on the type and the yield of the photogenerated ROS. This should be a substantial consideration when designing new efficient PSs.



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Well known second generation PSs for PDT are tetrapyrrole macrocycles, which include porphyrins15-21,

dihydroporphyrins

(chlorins)15-21

and

symmetric

tetrahydroporphyrins

(bacteriochlorin or BC as commonly used biochemical abbreviation)21-31,33-35. They are characterized by their low dark toxicity, the ease of tuning their photophysical properties and the ease of their functionalization with respect to the therapeutic applications. In comparison to other members of the tetrapyrrole family, BCs possess intense absorption bands (~ 1×105 dm3 mol-1 cm-1) located in the near infrared region of 720-850 nm, where porphyrins and chlorins do not absorb effectively. Indeed, BCs have long-lived triplets, high triplet quantum yields, and their triplet energies lie between ~104.6 and ~125.5 kJ mol-1.21 Therefore, they are able to generate 1

O2 and radicals with large quantum yields. These properties make BC a promising candidate for

PDT applications. Synthetic unsubstituted37 (u-BC, see Figure 1) and naturally occurring30 BCs are not feasible for PDT applications since they were readily susceptible to autooxidation. Structural modifications of the BC macrocycle via chemical substitution and/or the introduction of a metal into the molecule’s central cavity was found to improve their stability and water solubility and enhance the yields of 1O2 formation, as well as increasing their ability to localize with high selectivity to tumor tissue rather than healthy tissues.18-31 Furthermore, imparting hydrophilicity to BCs took place via using peripheral substituents having anionic and cationic groups such as sulfonate, carboxylate, and quaternary ammonium (see Figure 1). Currently,


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metal free (mTHPBC)38 and metal containing BCs (WST-09 and WST-11)35,39 have been chosen in clinical trials of PDT for different types of cancers.

Figure 1. Evolution of structures of unsubstituted porphyrin, chlorin and bacteriochlorin (u-BC) together with its derivatives and THPTS.

A new synthetic bacteriochlorin derivative (THPTS) with negligible dark toxicity is currently used as a potent photosensitizer for PDT.27,40-44 The results in vivo and in vitro revealed that THPTS exhibits a high photodynamic efficiency initiated by 760 nm photons excitation in animal C26 colon carcinoma and could achieve deep tumor-tissue penetration (1 cm).27,43



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Moreover, THPTS shows high efficiency as a potent antimicrobial photosensitizer.42,43 The tetracationic THPTS is highly efficient in the photoinactivation of both Gram-positive and Gramnegative bacteria. In the present study we investigate the influence of the cationic N-methylpyridinium substituents of THPTS; compared to the unsubstituted 7,8,17,18-tetrahydroporphyrin (u-BC), on the structural characteristics, photophysical and photochemical properties, solubility, and mechanistic features. This was carried out by using steady-state absorption, emission and phosphorescence, time-resolved spectroscopes, EPR spectroscopy with spin-traps. We also shed light onto the direct detection of the localization of THPTS in vivo by using fluorescence lifetime imaging microscopy (FLIM). Characterization of molecular orbitals and elucidation of different energy transfer efficiency from THPTS(T1) to molecular oxygen in aqueous and acetonitrile solutions were carried out by density functional theory (DFT) calculations. Our study shows that the balance between Type I and Type II processes is generally controlled by the photosensitizer structure as well as the photosensitizer environment. We show here neither 1O2 production nor the direct formation of other ROS (O2•― and •OH) from the excited triplet state of the PS is necessarily prerequisite for cell toxicity in PDT. This finding may influence the design of new efficient PSs for future PDT applications.

Experimental Section Materials. 5,10,15,20-tetrakis-(1-methyl-3-pyridyl)-21H,23H–7,8,17,18-tetrahydroporphyrin tetratosylat (THPTS)45 (C72H70N8O12S4; molecular weight 1367.66 Da, purity ≥ 95 %) was donated from TetraPDT GmbH, Rackwitz/Germany, and used as received (see Figure 2). Deionized water was taken from a milli-Q plus ultrapure water system (Millipore). All other


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chemicals were purchased from commercial sources and were of highest spectroscopic grade and used as received. Steady State Absorption and Luminescence Studies. Absorption spectra between 250 and 830 nm were measured with a UV-2101 PC (Shimadzu) UV–Vis spectrometer. Fully corrected fluorescence spectra were recorded on a FluoroMax-2 spectrometer (Jobin Yvon-Spex). To avoid aggregation and inner-filter effects, the absorption was adjusted to 0.05 at the excitation wavelength. Fluorescence quantum yields were determined for diluted solutions by the comparative method46,47 using styryl 8 (LDS 751) in ethanol as standard (ФF = 0.028).48 The steady-state 1O2 phosphorescence measurements were performed using a Fluorolog3TCSPC spectrometer (HORIBA – Jobin Yvon). The spectrometer was equipped with a Symphony InGaAs array in combination with an iHR320 imaging spectrometer. A detailed description of the calculation of the triplet quantum yields form

1

O2 phosphorescence

measurements is given in the supporting information. Time-Resolved Fluorescence Spectroscopy. Fluorescence lifetimes of THPTS were measured with the Flourolog3-TCSPC in combination with a R3809U-58 MCP (Hamamatsu) and a N-405L laser diode (Horiba Jobin Yvon) exciting at 403 nm (≤ 200 ps FWHM). The corresponding lifetimes were obtained from the exponential fits including the deconvolution with the instruments response function (IRF). Transient Absorption Spectroscopy. Spectral and kinetic information of the S1-SN transitions of THPTS were obtained from femtosecond laser photolysis transient absorption measurements. The setup consists of an amplified Ti/sapphire laser system CPA-2101 femtosecond laser (Clark MXR–output: 775 nm, 1 kHz, 270 nJ/pulse and 150 fs pulse width) using a transient absorption pump/probe detection system (TAPPS Helios - Ultrafast Systems).



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Kinetic and spectrum of the THPTS(T1) were investigated by nanosecond laser photolysis. The solutions were photolysed with the output (760 nm) from a dye laser (ScanMate 2c – Lambda Physik) using styryl 8 (Radiant Dyes) as laser dye pumped with the second harmonics (532 nm) from a Nd:YAG laser (Quanta-Ray GCR-11, Spectra Physics). Pulse widths of < 8.5 ns and energies between 0.5 and 4.0 mJ/pulse at 760 nm were selected. The optical detection is based on a pulsed Xenon lamp (XBO 150, Osram), a monochromator (Spectra Pro 275, Acton Research), R955 photomultiplier tube (Hamamatsu Photonics) or a fast Si-photodiode and a 1 GHz digital oscilloscope (TDS 684 A, Tektronix). The laser power of every laser pulse was registered using a bypath with a fast Silicon photodiode. A more detailed description is reported elsewhere.49 The triplet extinction coefficient of THPTS was determined by singlet depletion method (see supporting information). Electron Paramagnetic Resonance (EPR). Spin trap experiments in conjunction with EPR spectroscopy were carried out to identify the 1O2 generated by irradiation of THPTS in aqueous and acetonitrile solutions using a 75 W xenon lamp and an optical band pass filter with a transparency range of 720 – 950 nm. 2,2,6,6-tetramethyl-4-oxo-piperidine (TEMP) reacts specifically with 1O2 to form the stable radical 2,2,6,6-tetramethyl-4-oxo-piperidine-N-oxide (TEMPO).50 The 14N (hfs) coupling is an indicator for the formed radical.51 For the identification of O2•― or •OH radicals, the spin traps 2,2-dimethyl-4-phenyl-2H-imidazol-1-oxide (DMPIO) and phenyl-N-tert-butylnitrone (PBN) were used. Whereas DMPIO reacts only with •OH,52 PBN is sensitive to both radicals.53,54 Due to the low solubility of these two spin traps in water a small amount of acetonitrile (7%) was added to the aqueous solution. The EPR spectra were recorded with a BRUKER EMX micro spectrometer at room temperature (295 K). The simulations were performed with the freeware program EasySpin.55


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Subcellular Localization by Fluorescence Lifetime Imaging. Rat bladder carcinoma AY27 cells were provided from Lorraine Cancer Institute, Nancy, France.56 The AY27 tumor cell line was derived from bladder carcinomas induced in male Fischer (F 344) rats (Charles River Laboratories, Wilmington, MA, USA), fed continuously with FANFT (0.2%).57 Cells were seeded onto collagen A (Biochrom, Berlin, Germany) coated glass coverslips and cultured for 24 hrs in RPMI-1640 (Biochrom) cell culture medium supplemented with 20% FCS (Biochrom) and 5% Penicillin/Streptomycin (Sigma-Aldrich, Steinheim, Germany). Cells were incubated for 2 hrs at 37°C in the dark with 200 nM MitoTracker® green FM, and LysoTracker® green FM (Invitrogen, Karlsruhe, Germany) + 200 µM THPTS. After washing 3-times with Ringer solution (pH 7.2; in mM: CaCl2 1.90, KCl 5.90, NaHCH3 14.40, MgCl21.20 mM, NaCl120.90, NaH2PO4 1.55, glucose 11.49, Hepes 4.20) the cells were ready for FLIM analysis. A detailed description of the fluorescence lifetime imaging microscopy is given in the supporting information. Quantum Chemical Calculations. Quantum chemical calculations were performed by Density Functional Theory (DFT) using the B3LYP method58,59 as implemented in the Jaguar program60 version 7.6. The structures of studied molecules were optimized at B3LYP/6-31(d) level. The frequency analysis was made on the structures optimized in water to obtain thermodynamic parameters such as zero point energy (ZPE), and Gibbs free energy (G) at 298 K. The reaction enthalpies (ΔH) and Gibbs free energies of reaction (ΔG) were calculated as the difference of the calculated total enthalpies H and Gibbs free energies (G) between the reactants and products respectively. The interactions between the molecule and the solvent were evaluated at the same level of theory by Jaguar’s Poisson-Boltzmann solver (PBF)61 which fits the field produced by the solvent dielectric continuum to another set of point charges. The electronic structure of studied molecules, namely molecular orbitals, Mulliken atomic charges, spin density



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distribution and energy of excited states were further analyzed using Gaussian 09.62 The electronic transition spectra were calculated using the Unrestricted Time Dependent (UTD DFT)63 method at B3LYP/6-31G(d) level as implemented in the Gaussian 09 package.

Results Photochemical studies Ground-State Studies. The absorption spectra of THPTS in acetonitrile and aqueous solutions at room temperature are shown in Figure 2A. In each of the solvents, THPTS exhibits a characteristic bacteriochlorin absorption spectrum with Soret (or B) bands in the near-UV (corresponding to the S0S2 transitions) maximizing at 349 nm (By, ε = 67200 dm3 mol-1 cm-1) and 373 nm (Bx, ε = 69400 dm3 mol-1 cm-1) together with Q-bands (corresponding to S0S1 transitions) maximizing at 516 nm (Qx, ε = 34000 dm3 mol-1 cm-1) nm and 761 nm (Qy, ε = 62000 dm3 mol-1 cm-1). The shoulder at 416 nm is attributed to traces of a chlorin by-product which is unavoidable and nearly unseparatable.27,40 However, the absorption spectra in both solvents are similar and are not affected by the solvent polarity. Indeed, the spectra obeyed Lambert-Beer law and did not show any aggregation up to 0.01 mmol dm-3 dye concentration. This means that THPTS is not aggregating in the concentration range used in this study. For comparison, u-BC displays an absorption spectrum in n-hexane with B-bands maximizing at ≈ 340 nm (By, ε ≈ 130000 dm3 mol-1 cm-1) and ≈ 360 nm (Bx, ε ≈ 130000 dm3 mol-1 cm-1) and Qbands maximizing at 486 nm (Qx, ε ≈ 60000 dm3 mol-1 cm-1) and 720 nm (Qy, ε ≈ 130000 dm3 mol-1 cm-1).37


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Figure 2. (A) Absorption, (B) emission (exc = 720 nm), and (C) fluorescence excitation (emission = 790 nm) spectra of THPTS in acetonitrile ([THPTS] = 6.7 mol dm-3, green) and aqueous ([THPTS] = 5 mol dm-3, black) solutions at room temperature.

Excited Singlet State Studies. The fluorescence spectra were studied upon excitation of THPTS at room temperature in acetonitrile and aqueous solutions at 720 nm, see Figure 2B. 13 Environment ACS Paragon Plus


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Independent of the used solvent, a florescence band with a maximum at 767 nm is observed. Moreover, excitation of the THPTS at 350, 375 or 516 nm (Supporting Information, Figure 1) revealed emission spectra exhibiting two main maxima centered at 767 nm and 660 nm which belong to THPTS(S1) and a by-product chlorin(S1), respectively.40 On the other hand, the fluorescence spectrum of the chlorin-by-product was selectively observed upon excitation at 420 nm (Supporting Information, Figures 1 and 2). The excitation spectra for the 790 nm emission of THPTS in both solvents revealed similar spectra to those observed by UV-Vis spectroscopy with the main characteristic four absorption bands, and did not show any band of the chlorin by-product maximizing at 420 nm, see Figure 2C. These results indicate that the chlorin by-product has no influence in the deactivation mechanism of THPTS(S1) upon photoexcitation at 760 nm. Indeed, the normalized fluorescence excitation and emission spectra intersect at ~ 763 nm, owing to the lowest excited singlet state energy value (E0-0) of ~ 159 kJ mol-1. Fluorescence Quantum Yield Determination (ФF). The ФF values of THPTS were determined for diluted acetonitrile and aqueous solutions using styryl 8 (LDS 751) in ethanol as standard (ФF=0.028).48 The estimated ФF values are 0.03 and 0.02 in acetonitrile and water (with an error of ±5%), respectively. Fluorescence Kinetics. Generally, the fluorescence decay kinetics is determined by all the relaxation processes competing for the excited singlet state of a molecule. Time-resolved fluorescence measurements give information about excited singlet state deactivations. The kinetic analysis of the traces of THPTS monitored at 750 nm has revealed a monoexponential decay function in both solvents (see Figure 3). The fluorescence lifetimes measured in acetonitrile and aqueous solutions are 2.9 ns and 2.3 ns (the error is ± 10%), respectively.


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Figure 3. Fluorescence decay curves of THPTS in argon saturated acetonitrile (green) and aqueous (black) solutions after excitation at 403 nm. The solid red lines are the exponential fittings.

Investigation of the S1-SN Transitions. Transient absorption pump–probe experiments in argon saturated acetonitrile and aqueous solutions were conducted and provide insights into the S1-SN transitions of the THPTS molecule. The excited singlet state is formed right after the laser pulse showing a broad transient absorption with maxima at 450, 500, 545, 660 and 1050 nm accompanied by transient bleaching bands maximizing at 465, 485, 520 and 760 nm (see Figure 4A) mirroring the ground state absorption. This transient absorption decays with a lifetime of 3.1 ns (acetonitrile) and 2.3 ns (water) within 10% accuracy (see Figure 4B) – well matching with the observed fluorescence lifetimes (vide supra) – into the triplet manifold (vide infra).



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Figure 4. (A) Femtosecond transient absorption spectra of THTPS in argon saturated aqueous solution; 1 ps (black), 10 ps (red), 100 ps (green), 1000 ps (blue) and 7500 ps (cyan) after excitation at 775 nm. (B) Corresponding time absorption profiles at 650 nm (green) and 1050 nm (black). The solid red line in (B) is the exponential fitting.

Excited Triplet State Studies. Nanosecond laser flash photolysis of THPTS in oxygen-free aqueous and acetonitrile solutions was carried out. In aqueous solution, for instance, the obtained transient absorption spectra with short time delays on the nanosecond time scale as well as long time delays exhibit three apparent absorption maxima (i.e. positive signals) at 300, 395 and 600 nm, accompanied by transient bleaching bands peaking at 350, 520, and 760 nm, which are mirroring the ground state absorption (see Figure 5A). Furthermore, the solvent change from water to acetonitrile (Supporting Information, Figure 3) did not cause any change in the shape or


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relative intensities of the transient absorption spectra, i.e. similar transient absorption spectra were observed in both solvents. Moreover, the decay monitored at the absorption maxima showed a first-order kinetic behavior with corresponding lifetimes of 70 s and 110 s in water (see Figure 5B) and acetonitrile (Supporting Information, Figures 3 and 4), respectively. In the presence of oxygen, the transient absorption lifetimes are substantially reduced to about 65 ns and 390 ns in acetonitrile and water, respectively (see Figure 5C). Because of this typical and expected behavior, together with the sensitization experiments given below, the transient is assigned to the first excited triplet state of THPTS(T1). Moreover, the oxygen quenching rate constant (kq) was estimated in both solvents using different oxygen concentrations resulting in kq equals 2.2×109 and 2.3×109 dm3 mol-1 s-1 (within ±5% accuracy) in acetonitrile and water, respectively, (Supporting Information, Figures 5 and 6). Figure 5B also shows a comparison of the time profile of recovery of THPTS at 350 nm in water, with the decay of the THPTS(T1) at 395 nm. The traces are mirror images of each other and their lifetimes in acetonitrile ( = 110 s) are longer than those in water ( = 70 s) (the values of  are within ±5% accuracy). Indeed, the observation of apparently isosbestic points in the transient absorption spectra of THPTS(T1) (see Figure 5A) indicated that the triplet relaxation occurs without chemical consequence. This means that no radicals or radical ions have been detected under our experimental conditions. The molecular extinction coefficients of the first excited triplet state (εT) were estimated from the transient absorption spectra obtained by direct photoexcitation of THPTS in water and acetonitrile using the singlet depletion method. The εT values in both solvents are comparable to each other and are 8900±500, 26000±500, and 14800±500 dm3 mol-1 cm-1 at 300, 395, and 600 nm, respectively.



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Figure 5. (A) Nanosecond transient absorption spectra obtained after laser photolysis of THPTS saturated with N2 in aqueous solution (5 mol dm-3) taken 8 (●), 80 (▲), and 210 (■) s after the pulse, respectively, (laser energy = 2.5 mJ). (B) Experimental time profiles of N2 saturated aqueous solution at 395 and 350 nm. (C) Decay dynamics of T-T absorption change at 395 nm with O2 in acetonitrile (green) and aqueous (black) solutions. The solid red lines in (B) and (C) are the exponential fittings.


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Triplet Energy Transfer Studies, 1O2 Detection and Triplet Quantum Yield Determination of THPTS (ФT). To characterize the THPTS(T1) we carried out photosensitization experiments with acceptors such as ß-carotene (ß-C, ET1 = 88 kJ mol-1) and methylene blue (MB, ET1 = 138 kJ mol-1).64 A 510 nm long pass filter was used to avoid degradation of ß-carotene by the analyzing light. Laser photolysis experiments of deaerated-acetonitrile solutions of THPTS in the presence of low concentrations of ß-C were carried out. This resulted in an acceleration of the decay of THPTS(T1) accompanied by ß-C(T1) formation at 527 nm, where no absorption of THPTS was monitored at this wavelength in the absence of ß-C - see Figure 6. The depletion of the ground state absorption of THPTS at 520 nm hindered the usual detection of ß-C(T1) at its absorption maximum (515 nm). In contrast a photosensitization experiment employing MB as a triplet energy acceptor with a characteristic transient absorption maximum around 420 nm for its excited triplet state was not observed. This indicates that THPTS(T1) is energetically not feasible to induce MB(T1). In other words, THPTS(T1) is energetically bellow that of MB(T1), i.e. 88 kJ mol-1 < E(THPTS(T1)) < 138 kJ mol-1.

Figure 6. Experimental time profiles of N2-saturated acetonitrile solutions of 6.7 mol dm-3 THPTS without (green) and with ß-C (black) after excitation at 760 nm (measurement = 527 nm, laser power = 2.5 mJ).



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For further characterizing the THPTS(T1) and for determining their ФT in both solvents we carried out sensitization experiments using energy transfer from a triplet THPTS(T1) molecule to molecular oxygen in oxygen-saturated solutions to generate singlet oxygen emission with a maximum at 1270 nm. Zinc naphthalocyanine (ZnNc) as a standard substance (the determination of the triplet quantum yield for ZnNc is decribed in the supporting information) was used here because its absorption spectrum in toluene exhibits two absorption maxima at 350 nm and 760 nm (data not shown). However, this method is only realized when the energy transfer from a triplet sensitizer molecule to molecular oxygen is the most efficient process and no other competitive quenching reaction(s) occurs. In acetonitrile, however, the singlet oxygen emission was detected (Supporting Information, Figure 7) and 0.75 ± 0.10 was determined as singlet oxygen quantum yield (Ф) of THPTS as derived from the linear fit of the plot of the integral of 1

O2 emission vs. THPTS absorption at 760 nm. Assuming that the energy transfer from

THPTS(T1) to oxygen has an efficiency close to unity, the triplet quantum yield Ф of THPTS is derived as ФФ 0.75. Surprisingly, the yield of 1O2 in aqueous solution under the same conditions in the presence of THPTS was not detected. This indicates that 1O2 quantum yield in aqueous solution is lower than < 0.05 which is the detection limit of the employed setup. EPR and Spin Trap Investigations of 1O2, O2•― and •OH. Figure 7 shows the X band EPR spectra of 0.75 mmol·dm-3 THPTS with 6.5 mmol·dm-3 TEMP in water before and during the radiation with xenon lamp. The triplet results of the interaction of the unpaired electron with the 14

N nucleus of the TEMPO radical (a0N=(45 ± 1) MHz, g0 = 2.0054 ± 0.0002) and shows the

production of a very small amount of 1O2 during the irradiation. The same measurement in acetonitrile (0.10 mmol·dm-3 THPTS with 1.0 mmol·dm-3 TEMP) showed the same behavior with identical EPR parameters. The integration of the signals indicates a much higher yield of


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TEMPO radical in acetonitrile than in water. This is in a good agreement with the results of the optical spectroscopy.

Figure 7. X band EPR spectra of THPTS and TEMP in water (A) during radiation, (B) simulation, and (C) before irradiation.

The spin trap experiments with DMPIO and PBN was performed to identify the formation of O2•― or •OH radicals, respectively, after photolysis of THPTS. All samples were EPR silent, before and after irradiation. An increase of the spin trap concentration up to 100 mmol dm-3 displayed no existence of any O2•― or •OH radicals. This demonstrates the impossibility of the generation of O2•― or •OH in the presence of THPTS neither in acetonitrile nor in water.



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Figure 8. AY27 intracellular fluorescence colocalization (all images 30×30µm²): (A) confocal MitoTracker® green intensity image, (B) THPTS FLIM image, fluorescence lifetime τ1 colour coded from 300 to 800ps, (C) confocal LysoTracker® green intensity image, (D) THPTS FLIM colocalized with LysoTracker® green, (E) Fluorescence decay curve of the THPTS in organelles (black, taken at the position indicated by the white arrow in (B)) with the corresponding fitting (solid red line).

Fluorescence Imaging Studies in vivo. In an additional set of experiments we have imaged the localization of THPTS in living cells via fluorescence lifetime imaging microscopy (FLIM) together with colocalization experiments performed with organelle labeling dyes. The results revealed the accumulation of THPTS occurs mainly in the lysosomes of rat bladder carcinoma


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cells. Figure 8 shows the comparison of the THPTS-FLIM images (8B and 8D, excited at 750 nm, short time component false color coded from red (300 ps) to blue (800 ps) with the fluorescence intensity images (excited at 405 nm, grayscale images 8A and 8C) of MitoTracker® green and LysoTracker® green, respectively. The actual decay of the THPTS fluorescence follows a two exponential behavior. The short component with lifetimes between 150 and 400 ps is most pronounced at the lysosomes (see red colored features in the FLIM image Figure 8B) implying strong interaction within these organelle. The long component of 2.1 ns, which is similar to the fluorescence lifetime of THPTS in free aqueous solution, contributes only marginally to the signal intensity (see Figure 4). Both, 405 nm and 750 nm excitation of the cells without THPTS as the reference experiment displayed negligible autofluorescence. Quantum Chemical Calculations. Figure 9 shows structure and electron density distribution for the HOMO, HOMO-1, LUMO and LUMO+1 MOs. It indicates that the plane of the four substituted pyridyl rings is nearly perpendicular to the plane a bacteriochlorine ring (Supporting Information, Figure 8). In fact, the HOMO-1, HOMO, and LUMO MOs have a similar πsymmetry, and they are delocalized in the plane of a bacteriochlorine ring, whereas the LUMO+1 and the higher LUMO’s up to LUMO+4 (not shown) MOs have different kind of π-symmetry and they are delocalized in the plane of pyridyl rings. The calculations also indicated that the largest contribution to Q excitation arises from HOMO to LUMO and to LUMO+1 transitions, while the B excitation arises from HOMO-1 to LUMO and to LUMO+1 transitions. However, the wave function of the S1 state is comprised of 65% of the HOMO-LUMO configuration with oscillator strength of 0.24.



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Figure 9. Electron-density distribution of molecular orbitals for THPTS in the most stable structure as calculated at B3LYP/6-31 G(d) level.

Figure 10A demonstrates the calculated electronic transition spectra of THPTS in the gas phase and in solutions. The results reveal an essential influence of the polar solvents on energies of the excited states of THPTS in comparison with those of the gas phase. However, there is practically no difference between absorption spectra in acetonitrile and water. These findings are in good agreement with our experimental results. Furthermore, the calculated excited singlet and triplet states of THPTS in both solvents are 178.5 kJ mol-1 ( 1.85 eV) and 93.6 kJ mol-1 (0.97 eV), respectively. Indeed, the energies of THPTS(T1) were calculated in both solvents as energy difference

between

ground

optimized

singlet

and

triplet

states

at

B3LYP/6-

311+G(d,p)/PBF//B3LYP/6-31G(d) level of theory. The calculated zero point corrected energies of the T1-state are 97.5 and 94.5 kJ mol-1 in acetonitrile and water, respectively, which are close to 93.6 kJ mol-1 obtained from calculations of energy of excited states. Also the calculated H values of the possible electron transfer from THPTS(T1) to oxygen are +74 kJ mol-1 and +83 kJ mol-1 in acetonitrile and water, respectively.


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Figure 10. Absorption spectra in gas phase (solid black line), acetonitrile (solid red line) and water (dashed blue line) of THPTS and TMPyP4 calculated with TD B3LYP/6-31G(d)//PBF method.

Discussion Several studies revealed that cationic porphyrin derivatives are attractive agents for PDT applications because they photoinduce inactivation of tumor cells and microorganisms.65,66 Their photoactivity occur predominantly through Type II photoreaction process and their photocytotoxic effect is proportional to the overall positive charge of the molecule. These studies also revealed that positively charged sensitizers are better localized than negatively or neutral analogues in certain target sites. Some other reports indicated that tetracationic meso-tetra(4-Nmethylpyridinium)porphyrin (TMPyP4), for instance, appears to be a selective tumor localizer and possess antitumour activity upon irradiation by red light.67-70



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From this point of view, understanding the influence of the photophysical and chemical characteristics of the sensitizer that can be manipulated via chemical modifications is important for designing potent PSs. So, the goal of our study was to elucidate the influence of the four positively charged N-methylpyridinium substituents in THPTS on the molecular orbital characteristics, photophysical and photochemical properties, as well as mechanistic features. Here we discuss the impact of the net charge and position of the peripheral substituents in THPTS, compared to the unsubstituted 7,8,17,18-tetrahydroporphyrin, on the molecular orbital characteristics which in turn influence the photophysical properties. Also we discuss the distribution of THPTS in rat bladder caricinoma AY27 cells. Generally, the longest wavelength transition (Qy) is of particular interest because its excitation produces the lowest excited S1-state, which almost entirely controls the photophysical features. At first sight, the electron-donating substituents of THPTS - compared with unsubstituted bacteriochlorin (u-BC)- cause a significant red shift in all absorption maxima of THPTS particularly in the Qy-band (~ 40 nm). Also it should be noted that the four positively charged Nmethylpyridinium substituents of THPTS apparently cause a strong lowering in the energies of the frontier MOs, compared to those of u-BC (Supporting Information, Figure 9). The calculated excitation energy (ES1 = 206.5 kJ mol-1) of u-BC agrees with observed in experiment blue shift relative THPTS(S1). Furthermore, the HOMO energy of u-BC is higher than that of THPTS indicating better electron acceptor characteristic of THPTS. However, in comparison to THPTS, there was no essential solvent effect obtained from quantum chemical calculations between acetonitrile and water for the S1 and T1 states of u-BC. It may be caused by lower solvation energy of neutral u-BC (37.7 kJ mol-1) in comparison with very large solvation energy (1632 kJ mol-1) of positive charged THPTS.


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According to our photophysical parameters (excited state lifetimes, quantum yields and fluorescence (kF = ФF/F) and radiationless rate constants (kISC = ФISC/F and kIC = 1[ФF+ФISC])), we find that the chemical substitution in THPTS results in a short-lived excited singlet state (F ~ 2-3 ns) and low fluorescence quantum yield in both solvents (ФF ~ 2-3%, kF ~ 9×106 s-1). The short fluorescence lifetime is accompanied with a rapid ISC of high quantum yield (ФISC ~ 75%, kISC ~ 3×108 s-1) to the long-lived triplet state; 110 s and 70 s in acetonitrile and water, respectively, together with low internal conversion of ФIC ~ 22% and kIC ~ 7×107 s-1. Indeed, our results show large differences between the experimental fluorescence lifetime (F) and the overall natural lifetime (0 = 1/kF). Hence, the vast majority of photoexcited THPTS molecules is deactivated radiationless. Concerning the efficient ISC process, its probability depends on the extent of the spin-orbit coupling as well as the energy gap between the states involved.71 The quantum chemical calculations revealed that the higher triplet state T2 (ET2 = 125.4 kJ mol-1) lies below and close to the S1-state (ES1 = 178.5 kJ mol-1). Indeed, the S1- and T1-states possess a similar MO configuration, whereas those of S1 and T2 states are different. In spite of the very small energy gap between T2- and S1-states (E = 53.1 kJ mol-1) as well as their different orbital symmetry, this leads to strong overlap between electronic and vibrational levels, i.e. a large Franck-Condon factor, and in turn to an efficient ISC transition. The oxygen quenching rate constants (kq) in oxygen saturated acetonitrile and aqueous solutions are equal (~ 2×109 dm3 mol-1 s-1), assuming the mechanism to be the same in both solvents. This is generally characteristic for a triplet excited state quenching by oxygen in solution.72,73 Regarding the photogeneration of 1O2 via energy transfer from THPTS(T1) to molecular oxygen, our experimental results show different effectivity of the photosensitization of



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1

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O2 in both solvents. Additionally, quantum chemical calculations reveal that the energies of the

THPTS(T1) in acetonitrile and water are close to that of 1O2 (94.5 kJ mol-1 / 0.98 eV). This means that THPTS(T1) can in principle produce 1O2 via an energy transfer process. The question is now: what is the reason of these different 1O2 efficiencies? The reason might be the different ability of the solvent to promote radiationless deactivation particularly for such low energies excited states (42-125 kJ mol-1).74-77 To realize this assumption, we compare the excited state energies of THPTS and a standard tetracationic porphyrin analogue (TMPyP4), which has a large quantum yield of 1O2 production (values of Ф in water and methanol are 0.74 and 0.78, respectively).73,78 First, the calculated absorption spectra of TMPyP4 in polar solvents and gas phase show similar trend to those of THPTS as shown in Figure 10B. Furthermore, the spectra of TMPyP4 in solutions agree well with the data in the literature.79 Second, comparing the excited states energies of THPTS and TMPyP4 in acetonitrile and water (see Figure11), one can suppose the possibility of the effective nonradiative deactivation of triplet energy of THPTS(T1) in water through a strong resonance with the intensive first overtone of O‒H valence vibration. Such a possibility does not exist in acetonitrile resulting in an effective direct energy transfer from THPTS(T1) to oxygen. Otherwise, the ability of TMPyP4(T1) to generate 1O2 can be explained through ineffective nonradiative deactivation of triplet energy through O‒H vibration in this case, because of only the weak third overtone is in resonance with TMPyP4(T1).


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Figure 11. Energy scheme of excited states and possible deactivation channels of excited triplet state of THPTS and TMPyP4 molecules calculated with TD B3LYP/6-31G(d)//PBF method.

Now the question arises can THPTS excites aromatic systems like adenosine, tyrosine or tryptophan at critical positions in enzymes via Type II mechanism. The energy transfer from excited triplet THPTS molecules to any aromatic acceptor targets is still unlikely to happen due to two reasons. On one hand, the relatively large difference between the first excited triplet energy levels of THPTS and those of the assumed acceptor molecules (ET1 ≈ 310-350 kJ mol-1)80 exclude e.g. the Dexter mechanism. On the other hand, a resonance (FRET) mechanism by Förster which is usually well established for the singlet spin system and in few cases for triplet too is also excluded because there is no overlap between emission and absorption spectra of donor and acceptor molecules, respectively, as already required for this type of mechanism. EPR spin trapping of free radicals during THPTS photoexcitation show neither generation of O2•― nor •OH radicals in both solvents. These results, together with the positive H estimated by quantum chemical calculations for electron transfer from T1-state of THPTS or TMPyP4 to oxygen (HTMPyP4(T1) = +106 kJ mol-1 in water), demonstrate that this reaction is strong



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endothermic and can be excluded. According to these findings, we propose here that a mechanism of action may take place via reduction rather than oxidation of the THPTS(T1). In this particular case, a reducing agent substrate is required to induce electron transfer to the THPTS(T1) which then passes the electron to molecular oxygen resulting in the generation of O2•― radicals and subsequently to •OH radicals or other ROS (see reactions 2a and 2b). This type of electron transfer process has already been proposed and speculated about elsewhere.31,32,35,81,82 Finally, intracellular action, localization and selectivity of tetrapyrrole-based PSs depend upon their net charge, structure, stability, and the balance between hydrophobicity and hydrophilicity.83 It was reported that lysosomes and mitochondria are the most likely target sites for these PSs. Understanding the local target sites of PSs is of crucial importance for employing such systems in photodynamic therapy. THPTS molecules, if accumulated in cell organelles, are present in two different “states” – free and bound THPTS. Most THPTS molecules seem to be localized in close intracellular “contact” and interaction, which leads to a significant change in their fluorescence lifetime properties. The drop of the S1 lifetime by one order of magnitude is likely caused by “contact quenching” of fluorescence from the S1, assuming a constant quantum yield of the triplet, a similar rate of internal conversion, and no further photochemical decay channel from S1. These results point towards a strong interaction at a specific site of action in the cell but not necessarily a molecular selectivity and site-specificity of the THPTS photosensitizer. It also shows that the sensitizer is able to easily penetrate the cell wall – possibly due to the pronounced positive charges. Accumulation mainly in the lysosomes of rat bladder carcinoma cells shows that the dye is not selectively acting on special sites of the cell but is digested in a natural way when cells are fed with the PS. Nevertheless, this does not affect its general ability to effectively kill cells after photochemical activation.


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Conclusions In the present contribution we explored the effect of chemical substitution on THPTS on the molecular orbital characteristics, photophysical and photochemical properties, as well as mechanistic features. The interplay of the energetic position of the electronic state relative to the oxygen molecule, the dynamics of excited state and the efficiency of ROS generation via Type I and/or. Type II processes are studied in detail with a number of different spectroscopic techniques. We showed that for the present system neither singlet oxygen nor other reactive oxygen species are directly produced in water. Nevertheless, the chemically modified tetrapyrrole photosensitizer possesses a high photodynamic efficiency in aqueous solution after irradiation by near infrared light.27,40-44 This indicate that 1O2 and the direct generation of other ROS (O2•― and •

OH) via electron transfer oxidation of PS(T1) by molecular oxygen is not essentially needed for

a PS in PDT applications in aqueous environments – which is believed to be prerequisite and essential for toxic action of PSs in PDT. Instead, the mechanism of action of THPTS in water takes place via a secondary electron transfer reaction from a reducing agent substrate to the THPTS(T1), which has already been proposed and speculated about elsewhere.31,32,35,77,78 This may stem from the radical anion of the PS or a subsequent reaction of it producing toxic species in a secondary reaction. These findings open new strategies for designing new efficient PSs with spectra in the near-infrared spectral region enabling large penetration depths still displaying large cell-toxicity. The reduction mechanism proposed here for THPTS(T1) is certainly intriguing and promising for fine tuning PDT-sensitizers, however, much more work has to be done in order to shed more light into this alternate reaction channel for photodynamic therapy.



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ASSOCIATED CONTENT Supporting Information: Descriptions of fluorescence lifetime imaging microscopy, triplet extinction coefficients and triplet quantum yields determination. Fluorescence emission and excitation spectra of THPTS in water. Laser photolysis transient absorption spectra obtained in acetonitrile. Decay dynamics of THPTS(T1) in water and acetonitrile. Decay dynamics of THPTS(T1) in the presence of different oxygen concentrations. Singlet oxygen phosphorescence from THPTS or ZnNc in acetonitrile. Front and side views of THPTS structure. Electron-density distribution of molecular orbitals for u-BC. Complete author names for references shortened by using et al. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author E-mail: [email protected], Telephone: +49-(0)3412352229, +49-(0)34197-39784/39770 Fax: +49-(0)341-9739779 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors thank Prof. Dr. D. M. Guldi (University Erlangen - Nürnberg) for interesting discussions and support, Dr. J. Malig (University Erlangen - Nürnberg) for the providing ZnNc samples, Prof. Dr. A. Pöppl (Faculty of Physics, University of Leipzig) for help and support during the measurements of the EPR spectra, Dr. M. A. D’Hallewin (Lorraine Cancer Institut, Nancy, France) for kindly providing the AY27 cells, and Dr. T. Claudepierre (University of Lorraine, France) for his valuable discussions. B. Abel and A. Kahnt gratefully acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG) via grant AB 63/14-1 and KA 3491/2-1. 32 Environment ACS Paragon Plus

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TOC Image

Structure of THPTS and its photodynamic pathways that occur during photodynamic action after light activation by 760 nm in aqueous solution.


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Chemical Modification of a Tetrapyrrole-Type ... - ACS Publications

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