Reduction of Muscle-Invasive Tumors by Photodynamic Therapy with Tetrahydroporphyrin-Tetratosylat in an Orthotopic Rat Bladder Cancer Model.

Photodynamic therapy (PDT) is a promising option for minimal-invasive treatment of bladder cancer. Efficacy of PDT in muscle-invasive urothelial cancer is still hampered by low tissue penetration of most photosensitizers due to short excitation wavelength. The novel light reactive agent tetrahydroporphyrin-tetratosylat (THPTS) is excitable at near-infrared (760 nm), allowing tissue penetration of up to 15 mm. Here, we established an orthotopic rat bladder cancer model and examined the effects of THPTS-PDT on tumor growth in vivo, and analyzed molecular mechanisms in vitro We examined pharmacokinetics and subcellular localization, and evoked cell death mode in cultured rat urothelial carcinoma cells (AY-27). We used female F344 Fischer rats for in vivo studies. Ten rats each were used for THPTS-PDT and light-only control. Bladders were evaluated by macroscopy and histology. Temperature-dependent THPTS uptake resulted in endosomal/lysosomal localization. PDT (0-50 µmol/L THPTS; 10 J/cm2) induced early onset of apoptosis leading to dose-dependent cytotoxicity in AY-27 cells. Single-time transurethral THPTS-PDT (100 µmol/L THPTS; 10 J/cm2) in F344 rats led to significant reduction of muscle-invasive tumor number (2/10 vs. 7/10 in controls) and total tumor volume (60% reduction) 2 weeks after PDT, while sparing healthy tissue. Here, we report for the first time effective tumor growth control by PDT in vivo THPTS is a promising new photosensitizer with the advantage of higher therapeutic depth and the potential of high-selective therapy in muscle-invasive urothelial cancer. This approach possibly allows minimal-invasive bladder preserving treatment of bladder cancer without systemic side effects.

How fast is the reaction of hydrated electrons with graphene oxide in aqueous dispersions?

Understanding the mechanism of the reduction of graphene oxide (GO) is a key-question in graphene related materials science. Here, we investigate the kinetics of the reaction of radiolytically generated hydrated electrons with GO in water. The electron transfer proceeds on the ns time scale and not on the ps time scale, as recently reported by Gengler et al. (Nat. Commun., 2013, 4, 2560).

Chemical modification of a tetrapyrrole-type photosensitizer: tuning application and photochemical action beyond the singlet oxygen channel.

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 ((1)O2) 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 (1)O2 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.

Kinetic studies of the reduction of [Co(dmgH)2(py)(Cl)] revisited: mechanisms, products, and implications.

We report on a mechanistic investigation regarding the reduction of [Co(III)(dmgH)2(py)(Cl)] (dmg = dimethylglyoxime) by several complementary techniques. The reduction of [Co(III)(dmgH)2(py)(Cl)] was initiated by either electrochemical, photochemical, or pulse radiolytical techniques, and the corresponding products were analyzed by ESI mass spectrometry. In addition, all of the rate constants for each step were determined. We have found solid experimental as well as theoretical evidence for the appearance of a dinuclear complex [Co(II)Co(III)(dmgH)4(py)2(H2O)2](+) to be the final product of reduction, implying the initially reduced form of [Co(III)(dmgH)2(py)(Cl)] undergoes a dimerization with the starting material in solution.

Shedding light into the detailed excited-state relaxation pathways and reaction mechanisms of thionaphthol isomers.

Nanosecond laser flash photolysis employing transient detection of emission and absorption in combination with pulse radiolysis and quantum theory has been employed to shed light into the kinetics, quantum yields, and mechanisms of the deactivation of the first excited singlet state of 1- and 2-thionaphthols (NpSH(S(1))). In contrast to thiophenols (ArSH(S(1))), the results revealed that the decay of the first excited singlet state of 1- and 2-thionaphthols (NpSH(S(1))) is governed by radiationless internal conversion (Φ(IC) = 0.29-0.46; 0.016-0.190) and intersystem crossing (Φ(ISC) = 0.14-0.15; 0.4-0.6), respectively, with pronounced S-H photodissociation (Φ(D) = 0.40-0.55; 0.35-0.40). Fluorescence as a deactivation channel plays a minor role (Φ(F) = 0.001-0.010; 0.010-0.034). Quantum chemical calculations helped in understanding the formation of naphthylthiyl radicals and rationalizing the differences in the efficiency of intersystem crossing of the 1- and 2-thionaphthol systems.

Coordination-driven self assembly of a brilliantly fluorescent rhomboid cavitand composed of bodipy-dye subunits.

The two sp(3) hybridized fluorine atoms of a Bodipy dye have been synthetically replaced with the linear donor ligand 4-ethynylpyridine (-C≡C-Py) to form a rigid and highly symmetrical 109.5° building block in which the fluorophore subunit is vertically aligned to the plane formed by the -C≡C-Py donors. Upon reaction of the above tecton with a 90° organoplatinum acceptor unit, an intensely fluorescent rhomboid cavitand was manifested in solution. In contrast to the vast majority of coordination-driven self-assembled chromophoric systems, the present one fully conserves the excellent photophysical properties of the parent Bodipy dye. These unique features of the present metallosupramolecular entity constitute a fascinating metal-to-ligand self-assembled prototype for building compact and intensely luminescent materials with host-guest capabilities.

Ionization of amino-, thio- and hydroxy-naphtalenes via free (unhindered) electron transfer.

The electron transfer from various monosubstituted naphthyl derivatives (naphtols, NpOH; naphtylamines, NpNH2; and thionaphtols, NpSH) to parent n-BuCl radical cations was studied by means of pulse radiolysis. The experiments reveal the synchronous and direct formation of two types of transients: the metastable solute radical cation (NpXH(*+), X = heteroatom) and the corresponding heteroatom-containing radical (NpX(*)) in comparable amounts. This is explained in terms of the free (unhindered) electron transfer in nonpolar solvents, which is a bimolecular process reflecting femtosecond time scale events of intramolecular dynamic motions accompanied by significant changes of the electron distribution within the donor molecule.

Deactivation of the first excited singlet state of thiophenols.

On the bases of picosecond and nanosecond laser flash photolysis with detection by emission and absorption spectroscopy, a quantitative description is given of all deactivation channels of the first excited singlet state of thiophenols ArSH(S(1)) such as fluorescence, intersystem crossing (ISC), chemical dissociation into radicals, and radiation-less internal conversion (IC). For this purpose, the photolysis of thiophenol and its methyl-, methoxy-, and chloro-substituted derivatives was studied in solvents of increasing polarity: 1-chlorobutane, ethanol, and acetonitrile. The fluorescence lifetime of the thiophenols was found to range from some hundreds of picoseconds up to a few nanoseconds, correlating with fluorescence quantum yields between 0.001-0.040, at room temperature. Depending on the substitution pattern of the aromatic ring, the quantum yield of the S-H bond dissociation was found to be between 0.3-0.5, irrespective of the solvent polarity. In laser photolysis, no triplet formation of the investigated compounds could be observed neither by the direct way nor by subsequent sensitization with beta-carotene. As a difference to the total, the radiation-less internal conversion (Phi(IC)>or= 0.5) was found to be the dominating process.

Free electron transfer from xanthenyl- and fluorenylsilanes (Me3 or Ph3) to parent solvent radical cations: effects of molecule dynamics.

Parent radical cations of nonpolar solvents (alkanes and alkyl chlorides) ionize 9-(trimethylsilyl)xanthenes and 9-(trimethylsilyl)fluorenes in a diffusion-controlled electron transfer. The actual electron jump as the deciding part of the process does not require a defined encounter complex, and therefore the reactants are not subjected to any geometry optimization. Considering the molecule dynamics of the donors, bending motions of the silyl group are concerted with fluctuations of the highest occupied molecular orbital electrons. Ionizing such a standing conformer mixture creates metastable (microsecond) as well as dissociative donor radical cations. A mobility restriction of the benzylic silane group in positions vertical to the phenyl plane stabilizes the radical cations and accounts for a declining amount of dissociative radical cations, which undergo C-Si bond fragmentation in the order benzylsilane > xanthenylsilane > fluorenylsilane.

Improved pharmacokinetics, biodistribution and necrosis in vivo using a new near infra-red photosensitizer: tetrahydroporphyrin tetratosylat.

The search for better photosensitizers for photodynamic therapy of malignancies has led to the investigation of a new water-soluble, positively charged, and chemical stable tetrahydroporphyrin tetratosylat (THPTS) with a strong absorption at 760.5 nm, belonging to the bacteriochlorophyll family. THPTS undergoes a rapid uptake by human choroidal melanoma (CM) cells with a weak dark toxicity after a 24-h incubation (LD10 = 150 microM, LD50 = 6.0 mM). In response to laser light at 760+/-3 nm and doses of 10, 15 and 30 J/cm2, around 71%, 76%, and 92% of the CM cells were killed, respectively. Studies of pharmacokinetics and biodistribution in vivo (living mice) and ex vivo (excised organs) were made in a Balb/c mice bearing subcutaneously inoculated C26 colon carcinoma using fiber-optic spectrofluorimetry (FOS). Tumours were irradiated 3 h after intraperitoneal (i.p.) injection of 5.0 mg/kg THPTS with an incoherent light source at 750+/-20 nm and an intensity of 100 mW/cm2 and fluences of 60, 90 and 120 J/cm2. THPTS demonstrated preferential accumulation in C26 colon carcinoma in comparison with most normal tissues except kidneys. For the tissues of liver, colon, muscle, and spleen the tumour/normal tissue ratio (TNTR) ranged from 8.0 to 50. After irradiation with 120 J/cm2 the depth of tumour necrosis reached 15 mm. Histological examination of the tumour samples 24 h after THPTS-PDT, revealed severe stasis in the blood vessels and coagulative necrosis. These results suggest that THPTS-PDT may be of particular importance in the treatment of accessible malignancies.

Ionization of aniline and its N-methyl and N-phenyl substituted derivatives by (free) electron transfer to n-butyl chloride parent radical cations.

The electron transfer from aniline and its N-methyl as well as N-phenyl substituted derivatives (N-methylaniline, N,N-dimethylaniline, diphenylamine, triphenylamine) to parent solvent radical cations was studied by electron pulse radiolysis in n-butyl chloride solution. The ionization results in the case of aniline (ArNH2) and the secondary aromatic amines (Ar2NH, Ar(Me)NH) in the synchronous and direct formation of amine radical cations, as well as aminyl radicals, in comparable amounts. Subsequently, ArNH2*+ deprotonates in a delayed reaction with the present nucleophile Cl-, and forms further ArNH*. In contrast, tertiary aromatic amines such as triphenylamine and dimethylaniline yield primarily the corresponding amine radical cations Ar3N*+ or Ar(Me2)N*+, only. The persistent Ar3N*+ forms a charge transfer complex (dimer) with the parent amine molecule, whereas Ar(Me2)N*+ deprotonates to carbon-centered radicals Ar(Me)NCH2*.

Quantitative description of the deactivation channels of the first excited singlet state of 2- and 4-thiosalicylic acids.

On the bases of picosecond and nanosecond laser flash photolysis with detection by emission and absorption spectroscopy, a quantitative description is given of all of the deactivation channels of the first excited singlet state of 2- and 4-thiosalicyclic acids (TS) such as fluorescence, intersystem crossing (ISC), chemical dissociation into radicals, and radiationless internal conversion (IC). For this purpose, the investigated compounds were studied in solvents of increasing polarity: 1-chlorobutane, acetonitrile, ethanol, and water. As an exception for thiols, and in contrast to recent findings on thiophenol and its methyl- and methoxy-substituted derivatives, the photoinduced first excited triplet state of the thiosalicyclic acids was directly observed by its T1-T(n) absorption spectra and characterized by sensitization experiments. According to the direct determination of the quantum yields of all deactivation channels of the first excited singlet state of 2- and 4-thiosalicylic acid isomers, it was found that the compounds studied have small fluorescence quantum yields (phi(F) = 0.003-0.009) and higher ones for triplet formation (phi(T) = 0.10-0.35) and S-H photodissociation (phi(D) = 0.05-0.15). As a difference to the total, the radiationless internal conversion (phi(IC) = 0.6-0.8) was found to be the dominating process.

Ionization of cyclic aromatic amines by free electron transfer: products are governed by molecule flexibility.

The bimolecular electron transfer from secondary aromatic amines to parent radical cations of nonpolar solvents such as alkanes and alkyl chlorides results in the synchronous formation of amine radical cations as well as aminyl radicals, in comparable amounts. If as for cyclic aromatic amines (c-Ar(2)NH) the intramolecular bending motion around the amine group is restricted in varying degrees (acridane, phenothiazine) or completely prevented (carbazole), then this picture is modified. In the free electron transfer, the completely rigid carbazole yields exclusively amine radical cations. Acridane exhibits preferred radical cations, but phenothiazine with the more flexible six-membered ring involving sulfur as a further heteroatom follows the common two-product rule; see above. The phenomenon is reasoned by a peculiarity in the bimolecular free electron transfer where after diffusional approach the actual electron jump proceeds in the ultrashort time range. Therefore, it reflects femtosecond molecular motions which, in the case of free mobility, continuously pass through different molecule conformers, combined with fluctuation of the electrons of the responsible molecular n-orbitals. The rigid systems, however, do not show this effect because of a nonexistent bending motion.

Thiyl radical interaction with pyrimidine C5-C6 double bond.

Addition and elimination interaction of thiyl radicals with the C5-C6 double bond in pyrimidines was studied by the pulse radiolysis technique in aqueous solution with the use of different monitoring systems. For this purpose, p-thiocresol, cysteamine hydrochloride, and mercaptoethanol were used. The rate constants of addition and elimination of thiyl radicals were determined by applying the modified version of ACUCHEM (computer program for modeling complex reaction systems). Aliphatic thiyl radicals add to the pyrimidine C5-C6 double bond with k = 1.0-3.0 x 10(7) dm3 mol(-1) s(-1), whereas elimination takes place with k = 0.7-2.0 x 10(5) s(-1). Quantum chemical calculations at the B3LYP/6-31G(d)/PCM level show that the addition should occur at the C6 position of the pyrimidine ring and that the energy of interaction between thiyl radicals and the pyrimidine double bond C5-C6 is low.

Novel bacteriochlorine for high tissue-penetration: photodynamic properties in human biliary tract cancer cells in vitro and in a mouse tumour model.

Photodynamic therapy of bile duct cancer using hematoporphyrin derivative (HPD) and laser light of 630 nm wavelength is confined to a tumouricidal tissue penetration of 4 mm, which might be doubled with laser light between 700 and 800 nm. Therefore, we investigated the photosensitising properties of a novel bacteriochlorine, tetrakis-pyridyl-tetrahydroporphyrin tosylat (THP) with high absorption at 763 nm. Two biliary cancer cell lines (BDC, GBC) were incubated with HPD or THP to assess cellular uptake kinetics, dark cytotoxicity, and photodynamic cytotoxicity (laser light exposure 1-20 J/cm2). Tumours grown from BDC cells in subcutaneous tissue of severe combined immunodeficient mice were treated with laser light of 30 J/cm2 after injection of THP. The concentrations that killed 50% of cells in the dark were 680 microg/ml of HPD, but > 6400 microg/ml of THP in BDC cells, and 220 microg/ml of HPD, but 6400 microg/ml of THP in GBC cells. Both cell lines exhibited uptake and retention of THP and photodynamic cytotoxicity (up to 86% cells killed). THP induced tumour-selective phototoxicity in the cholangiocarcinoma model. The novel bacteriochlorine THP exhibits photosensitiser properties in biliary tract cancer cells in vitro and in vivo and could achieve deep tumouricidal tissue penetration due to photoactivation at 763 nm.