Ulrich Schatzschneider

Cellular Uptake, Cytotoxicity, and Metabolic Profiling of Human Cancer Cells Treated with Ruthenium(II) Polypyridyl Complexes [Ru(bpy)2(N-N)]Cl2 with N-N = bpy, phen, dpq, dppz, and dppn

A series of five ruthenium(II) polypyridyl complexes [Ru(bpy)2(NN)]Cl2 was tested against human HT‐29 and MCF‐7 cancer cell lines. Cellular uptake efficiency and cytotoxicity were found to increase with the size of the aromatic surface area of the NN ligand. The most active compound carrying the dppn ligand exhibits a low micromolar IC50 value against both cell lines comparable to that of cisplatin under similar conditions. Continuous measurement of oxygen consumption, extracellular acidification rate, and impedance of the cell layer with a chip‐based sensor system upon exposure to the complexes showed only small changes for the first two parameters throughout the series. A significant and irreversible decrease in impedance was, however, found for the dppn compound. This suggests that its biological activity is related to modifications in cell morphology or cell–cell and cell–matrix contacts.

Dimerization of a cell-penetrating peptide leads to enhanced cellular uptake and drug delivery

Summary Over the past 20 years, cell-penetrating peptides (CPPs) have gained tremendous interest due to their ability to deliver a variety of therapeutically active molecules that would otherwise be unable to cross the cellular membrane due to their size or hydrophilicity. Recently, we reported on the identification of a novel CPP, sC18, which is derived from the C-terminus of the 18 kDa cationic antimicrobial protein. Furthermore, we demonstrated successful application of sC18 for the delivery of functionalized cyclopentadienyl manganese tricarbonyl (cymantrene) complexes to tumor cell lines, inducing high cellular toxicity. In order to increase the potential of the organometallic complexes to kill tumor cells, we were looking for a way to enhance cellular uptake. Therefore, we designed a branched dimeric variant of sC18, (sC18)2, which was shown to have a dramatically improved capacity to internalize into various cell lines, even primary cells, using flow cytometry and fluorescence microscopy. Cell viability assays indicated increased cytotoxicity of the dimer presumably caused by membrane leakage; however, this effect turned out to be dependent on the specific cell type. Finally, we could show that conjugation of a functionalized cymantrene with (sC18)2 leads to significant reduction of its IC50 value in tumor cells compared to the respective sC18 conjugate, proving that dimerization is a useful method to increase the drug-delivery potential of a cell-penetrating peptide.

Label‐Free Imaging of Metal–Carbonyl Complexes in Live Cells by Raman Microspectroscopy

The search for novel metal complexes with therapeutic activity, in particular against cancer and infectious diseases, is an active and important area of research in medicinal inorganic chemistry. In addition to the well-studied platinumand ruthenium-based coordination complexes, organometallic compounds have gained considerable importance in recent years. Of those, metal–carbonyl compounds are steadily increasing in interest, with some exhibiting remarkable antitumor activity. The most prominent example is probably the use of such compounds as “solid storage forms” for carbon monoxide. These CO-releasing molecules (CORMs) allow the biological action of this important small molecule messenger to be investigated. To elucidate the biological mode of action of any drug candidate it is mandatory to obtain a detailed picture of the intracellular distribution of such substances and how it evolves over time. Until now, the localization of metal complexes inside cells has been studied using X-ray fluorescence (XRF) 14] and atomic absorption spectroscopy (AAS). While AAS offers high sensitivity but almost no spatial resolution, XRF requires intense X-ray sources such as synchrotrons which will cause damage to biological tissue and is also not routinely available as an analytical technique. Most cellular studies therefore use fluorescence microscopy. Furthermore, this technique requires the additional attachment of a fluorescent label, which might be difficult. Optical excitation can also cause additional problems such as the onset of photochemical reactions or photobleaching. Moreover, the label can alter the biodistribution and properties of the molecule of interest, as recently shown for ruthenium–bipyridyl complexes. Efforts have been made to overcome these limitations by identifying biologically active metal complexes which show inherent fluorescence in vivo, but this has only been possible for a small number of metal–ligand combinations. Thus, it is highly desirable to develop innovative and generally applicable imaging techniques for the study of the uptake and distribution of bioactive metal complexes which do not require any labeling or special photophysical properties but instead use the intrinsic spectroscopic signature of the compound of interest. Raman microspectroscopy is emerging as a powerful noninvasive method to assess and image cellular compartments and processes without further sample preparation or labeling. Since Puppels et al. first showed the feasibility of confocal Raman microspectroscopy for imaging cells, its ability to study whole cells and subcellular organelles such as the nucleus and chromatin, mitochondria, and lipid bodies has been demonstrated by various research groups. Apart from imaging subcellular features, Raman imaging has been used to follow the uptake of molecules by cells. So far, however, these investigations have been restricted to the incorporation of deuterated building blocks as sensitive and specific markers into bio(macro)molecules. Herein, we investigate the uptake and cellular distribution of the new manganese-based CORM [Mn(tpm)(CO)3]Cl (tpm = tris(1-pyrazolyl)methane), which has photoinduceable cytotoxic activity against cancer cells. Metal–carbonyl complexes such as [Mn(tpm)(CO)3]Cl show strong C O stretching vibrations between 1800 and 2200 cm , a region where vibrational signals from the constituents of the cell are negligible. We show that the C O vibrations of this compound can be used as an ideal marker for imaging these complexes in living cancer cells. Although the spectroscopic signature of metal–carbonyl compounds has already been used in bioanalytical techniques such as the carbonyl–metal immunoassay (CMIA), their use in cellular imaging is so far unprecedented, except for an investigation of osmium–carbonyl clusters in dried cells by using infrared microscopy. The IR and Raman spectra of solid [Mn(tpm)(CO)3]Cl show strong C O stretching vibrations at about 1944 and 2050 cm , as expected for local C3v symmetry (Figures S1 and S2 A in the Supporting Information). The different relative intensities of the two peaks can be explained by the distinct selection rules for Raman and IR spectroscopy. The O H stretching vibration localized at about 3400 cm 1 dominates the spectrum of a 2 mm aqueous solution of [Mn(tpm)[*] K. Meister, Dr. D. A. Schmidt, Prof. Dr. M. Havenith Lehrstuhl f r Physikalische Chemie II, Ruhr-Universit t Bochum Universit tsstrasse 150, 44801 Bochum (Germany) E-mail: martina.havenith@rub.de Homepage: www.rub.de/pc2

Acyloxybutadiene Iron Tricarbonyl Complexes as Enzyme‐Triggered CO‐Releasing Molecules (ET‐CORMs)

Molecular hazardous materials transport: Enzyme-triggered CO-releasing molecules (ET-CORMs) offer new options for the delivery of CO. The cleavage of dienylester iron tricarbonyl complexes by an esterase under mild oxidative conditions generates CO, which causes a strong inhibiting activity of the compounds to inducible nitric oxide synthase as shown in a cellular assay.

DC and AC conductivity and dielectric properties of the metal-radical compound: Aqua[bis(2-dimethylaminomethyl-4-NIT-phenolato)]copper(II)

Abstract The AC and DC conductivity and dielectric properties of the crystalline metal-radical compound aqua[bis(2-dimethylaminomethyl-4-NIT-phenolato)]copper(II) have been investigated. The DC electrical measurements show that the compound is a typical semiconductor with moderate activation energy E a =0.56 eV and at room conductivity σ 25 =1.38×10 −6 S cm −1 as its electrical conductivity increases with increasing temperature. The AC conductivity of the sample is found to be proportional to ωs. The temperature dependence of both the AC conductivity and the frequency exponent s is reasonably well interpreted in terms of the correlated barrier hopping model. The dielectric properties have been investigated as a function of frequency and temperature. Values of the dielectric constant e′ and dielectric loss e″ were found to decrease with frequency and increase with temperature. The effect of different temperatures on the AC conductivity and dielectric properties was also investigated. The AC conductivity and values of the dielectric constant increase with increasing temperature.

Next generation PhotoCORMs: polynuclear tricarbonylmanganese(I)-functionalized polypyridyl metallodendrimers.

The first CO-releasing metallodendrimers, based on polypyridyl dendritic scaffolds functionalized with Mn(CO)3 moieties, of the general formula [DAB-PPI-{MnBr(bpy(CH3,CH═N))(CO)3}n], where DAB = 1,4-diaminobutane, PPI = poly(propyleneimine), bpy = bipyridyl, and n = 4 for first- or n = 8 for second-generation dendrimers, were synthesized and comprehensively characterized by analytical (HR-ESI mass spectrometry and elemental analysis) and spectroscopic ((1)H, (13)C{(1)H}-NMR, infrared, and UV/vis spectroscopy) methods. The CO-release properties of these compounds were investigated in pure buffer and using the myoglobin assay. Both metallodendrimer generations are stable in the dark in aqueous buffer for up to 16 h but show photoactivated CO release upon excitation at 410 nm, representing a novel class of macromolecular photoactivatable CO-releasing molecules (PhotoCORMs). No scaling effects were observed since both metallodendrimers release ∼65% of the total number of CO ligands per molecule, regardless of the generation number. In addition, the mononuclear model complex [MnBr(bpy(CH3,CH═NCH2CH2CH3))(CO)3] was prepared and comprehensively studied, including DFT/TDDFT calculations. These metallodendrimer-based PhotoCORMs afford new methods of targeted delivery of large amounts of carbon monoxide to cellular systems.

Protease-activatable organometal-Peptide bioconjugates with enhanced cytotoxicity on cancer cells.

Over the past years, numerous promising new metalorganic lead structures have been developed exhibiting highly active cytostatic properties. However, the efficiency of such chemotherapeutics in the treatment of tumors is often limited by their low therapeutic index due to their short half-life, lack of tumor selectivity, and associated side effects. Furthermore, the membrane barrier often restricts their cellular uptake by passive diffusion. In this contribution, we describe the synthesis, cellular uptake, and biologic activity of a series of cymantrene-peptide conjugates. Cymantrene CpMn(CO)(3) is a robust organometallic group, which is stable in air and water and easy to functionalize. In this work, some new cymantrene derivatives with different linkers between the half-sandwich complex and the carboxylate group were attached to the cell-penetrating peptide sC18 that should act as a transporter for the metal moiety. All conjugates were characterized for their cytotoxic activity on human breast adenocarcinoma cells (MCF-7) and human colon carcinoma cells (HT-29). We found that bioconjugates bearing two cymantrene groups were more active than the monofunctionalized ones. By the introduction of a cathepsin B cleavage site next to the organometallic group, the biologic activity could be in increased even further. Fluorescence microscopy studies and apoptosis assays gave preliminary hints on the mode of action of these systems.

Iron Metal–Organic Frameworks MIL‐88B and NH2‐MIL‐88B for the Loading and Delivery of the Gasotransmitter Carbon Monoxide

Crystals of MIL-88B-Fe and NH2-MIL-88B-Fe were prepared by a new rapid microwave-assisted solvothermal method. High-purity, spindle-shaped crystals of MIL-88B-Fe with a length of about 2 μm and a diameter of 1 μm and needle-shaped crystals of NH2-MIL-88B-Fe with a length of about 1.5 μm and a diameter of 300 nm were produced with uniform size and excellent crystallinity. The possibility to reduce the as-prepared frameworks and the chemical capture of carbon monoxide in these materials was studied by in situ ultrahigh vacuum Fourier-transform infrared (UHV-FTIR) spectroscopy and Mössbauer spectroscopy. CO binding occurs to unsaturated coordination sites (CUS). The release of CO from the as-prepared materials was studied by a myoglobin assay in physiological buffer. The release of CO from crystals of MIL-88B-Fe with t(1/2) = 38 min and from crystals of NH2-MIL-88B-Fe with t(1/2) = 76 min were found to be controlled by the degradation of the MIL materials under physiological conditions. These MIL-88B-Fe and NH2-MIL-88B-Fe materials show good biocompatibility and have the potential to be used in pharmacological and therapeutic applications as carriers and delivery vehicles for the gasotransmitter carbon monoxide.

[N,N′-Bis(salicylidene)-1,2-phenylenediamine]metal complexes with cell death promoting properties

We developed N,N′-bis(salicylidene)-1,2-phenylenediamine (salophene, 1) as a chelating agent for metal ions such as Mn(II/III), Fe(II/III), Co(II), Ni(II), Cu(II), and Zn(II). The resulting complexes, from which owing to the carrier ligand a selective mode of action is assumed, were tested for antiproliferative effects on the MCF-7 breast cancer cell line. The cytotoxicity in this assay depended on the nature of the transition metal used. Iron complexes in oxidation states +II and +III (3, 4) strongly reduced cell proliferation in a concentration-dependent manner, whereas, e.g., the manganese analogues 5 and 6 were only marginally active. Therefore, the [N,N′-bis(salicylidene)-1,2-phenylenediamine]iron(II/III) complexes 3 and 4 were selected for studies on the mode of action. Both complexes possessed high activity against various tumor cells, for instance, MDA-MB-231 mammary carcinoma cells as well as HT-29 colon carcinoma cells. They were able to generate reactive oxygen species, showed DNA binding, and induced apoptosis. Exchange of 1 by N,N′-bis(salicylidene)-1,2-cyclohexanediamine (saldach, 2) yielding complexes 7 and 8 reduced the in vitro effects drastically. An unequivocal mode of action cannot be deduced from these results, but it seems to be very likely that cell death is caused by interference with more than one intracellular target.

Introducing [Mn(CO)3(tpa-κ3N)]+ as a novel photoactivatable CO-releasing molecule with well-defined iCORM intermediates – synthesis, spectroscopy, and antibacterial activity

[Mn(CO)3(tpa-κ(3)N)]Br was prepared as a novel photoactivatable CO-releasing molecule (PhotoCORM) from [MnBr(CO)5] and tris(2-pyridylmethyl)amine (tpa) for the delivery of carbon monoxide to biological systems, with the κ(3)N binding mode of the tetradentate tpa ligand demonstrated by X-ray crystallography. The title compound is a CORM prodrug stable in solution in the dark for up to 16 h. However, photoactivation at 365 nm leads to CO release from the metal coordination sphere and transfer to haem proteins, as demonstrated by the standard myoglobin assay. Different iCORM intermediates could be detected with solution IR spectroscopy and assigned using DFT vibrational calculations. The antibacterial activity of the complex was studied on Escherichia coli. No effects were observed when the cultures were either kept in the dark in the presence of PhotoCORM or illuminated in the absence of metal complex. However, photoactivation of [Mn(CO)3(tpa-κ(3)N)]Br at 365 nm led to the appearance of the spectral signatures of CO-coordinated haems in the terminal oxidases of the bacterial electron transport chain in whole-cell UV/Vis absorption spectra. Significant internalization of the PhotoCORM was demonstrated by ICP-MS measurement of the intracellular manganese concentration. In particular when using medium with succinate as the sole carbon source, a very pronounced and concentration-dependent decrease in the E. coli growth rate could be observed upon illumination in the presence of metal complex, which is attributed to the constrained energy metabolism under these conditions and a strong indicator of terminal oxidase inhibition by carbon monoxide delivered from the PhotoCORM.