Electrochemical, Computational, and Photophysical Characterization of New Luminescent Dirhenium–Pyridazine Complexes Containing Bridging OR or SR Anions

A series of [Re₂(μ-ER)₂(CO)₆(μ-pydz)] complexes have been synthesized (E = S, R = C₆H₅, <b>2</b>; E = O, R = C₆F₅, <b>3</b>; C₆H₅, <b>4</b>; CH₃, and <b>5</b>; H, <b>6</b>), starting either from [Re­(CO)₅O₃SCF₃] (for <b>2</b> and <b>4</b>), [Re₂(μ-OR)₃(CO)₆]⁻ (for <b>3</b> and <b>5</b>), or [Re₄(μ₃-OH)₄(CO)₁₂] (for <b>6</b>). Single-crystal diffractometric analysis showed that the two μ-phenolato derivatives (<b>3</b> and <b>4</b>) possess an idealized <i>C</i>₂ symmetry, while the μ-benzenethiolato derivative (<b>2</b>) is asymmetrical, because of the different conformation adopted by the phenyl groups. A combined density functional and time-dependent density functional study of the geometry and electronic structure of the complexes showed that the lowest unoccupied molecular orbital (LUMO) and LUMO+1 are the two lowest-lying π* orbitals of pyridazine, whereas the highest occupied molecular orbitals (HOMOs) are mainly constituted by the “t_2<i>g</i>” set of the Re atoms, with a strong Re–(μ-E) π* character. The absorption spectra have been satisfactorily simulated, by computing the lowest singlet excitation energies. All the complexes exhibit one reversible monoelectronic reduction centered on the pyridazine ligand (ranging from −1.35 V to −1.53 V vs Fc⁺|Fc). The benzenethiolato derivative <b>2</b> exhibits one reversible two-electron oxidation (at 0.47 V), whereas the OR derivatives show two close monoelectronic oxidation peaks (ranging from 0.85 V to 1.35 V for the first peak). The thioderivative <b>2</b> exhibits a very small electrochemical energy gap (1.9 eV, vs 2.38–2.70 eV for the OR derivatives), and it does not show any photoluminescence. The complexes containing OR ligands show from moderate to poor photoluminescence, in the range of 608–708 nm, with quantum yields decreasing (ranging from 5.5% to 0.07%) and lifetimes decreasing (ranging from 550 ns to 9 ns) (<b>3</b> > <b>4</b> > <b>6</b> ≈ <b>5</b>) with increasing emission wavelength. The best emitting properties, which are closely comparable to those of the dichloro complex (<b>1</b>), are exhibited by the pentafluorophenolato derivative (<b>3</b>)

Luminescent Conjugates between Dinuclear Rhenium Complexes and Peptide Nucleic Acids (PNA): Synthesis, Photophysical Characterization, and Cell Uptake

Different PNA decamers have been appended to a luminescent [Re₂(μ-Cl)₂(CO)₆(μ-1,2-diazine)] complex, to obtain conjugates suitable for cellular imaging. The new compounds can be dissolved in water in the presence of an amount of DMSO as small as 0.4–0.6 mol %. The conjugation with PNA did not perturb the photoluminescence behavior of the organometallic fragment: emission from ³MLCT excited states, centered at ca. 610 nm, was observed, with satisfactory photoluminescence quantum yields (Φ = ca. 0.01, in aerated water). Moreover, the emission could be stimulated also by two-photon excitation. Experiments of cell uptake, performed with different cell lines and under different experimental conditions, showed that the nature of the PNA oligomer strongly affects the biointeraction, while no major differences were observed among the cell lines investigated. The Re-PNA conjugates containing neutral PNA decamers (either homothymine or a standard sequence of the four nucleobases) showed a marked tendency to concentrate in the nuclear region, whereas nucleus penetration was more difficult for the free dirhenium complex. Staining of nucleus and cytoplasm with different colors was generally observed. No nucleus penetration was instead observed for a water-soluble Re-PNA homothymine decamer end-capped with four lysine residues, which localized in endosome-like compartments

A Luminescent Poly(amidoamine)–Iridium Complex as a New Singlet-Oxygen Sensitizer for Photodynamic Therapy

A polymer complex (<b>1</b>_<b>P</b>) was synthesized by binding bis­(cyclometalated) Ir­(ppy)₂⁺ fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly­(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir­(ppy)₂(bap)]⁺ (<b>1</b>_<b>M</b>, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution <b>1</b>_<b>P</b> gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to <b>1</b>_<b>P</b> having a photoluminescence quantum yield greater than that of <b>1</b>_<b>M</b> (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the ³MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O₂ with 1,5-dihydroxynaphthalene showed that <b>1</b>_<b>P</b> is able to sensitize ¹O₂ generation but with half the quantum yield of <b>1</b>_<b>M</b>. Cellular uptake experiments showed that both <b>1</b>_<b>M</b> and <b>1</b>_<b>P</b> are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both <b>1</b>_<b>M</b> and <b>1</b>_<b>P</b> are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for <b>1</b>_<b>M</b> was higher than that for <b>1</b>_<b>P</b> (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with <b>1</b>_<b>P</b> showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by <b>1</b>_<b>M</b> was avoided by binding the iridium sensitizers to the poly­(amidoamine)

Luminescent Rhenium and Ruthenium Complexes of an Amphoteric Poly(amidoamine) Functionalized with 1,10-Phenanthroline

A new amphoteric copolymer, <b>PhenISA</b>, has been obtained by copolymerization of 4-(4′-aminobutyl)-1,10-phenanthroline (BAP) with 2-methylpiperazine and bis­(acrylamido)­acetic acid (BAC) (6% of phenanthroline-containing repeating units). The copolymer showed excellent solubility in water, where it self-aggregated to give clear nanoparticle suspensions (hydrodynamic diameter = 21 ± 2 nm, by dynamic light scattering (DLS) analysis). The phenanthroline pendants of the polymer stably coordinated either Re­(CO)₃⁺ or Ru­(phen)₂²⁺ fragments, affording luminescent <b>Re-PhenISA</b>, <b>Re-Py-PhenISA</b>, and <b>Ru-PhenISA</b> polymer complexes, emitting from triplet metal-to-ligand charge transfer (³MLCT) excited states (with λ_em = 608, 571, and 614 nm, respectively, and photoluminescence quantum yields Φ_em = 0.7%, 4.8%, and 4.1%, in aerated water solution, respectively). DLS analyses indicated that the polymer complexes maintained the nanosize of <b>PhenISA</b>. All the complexes were stable under physiological conditions (pH 7.4, 0.15 M NaCl) in the presence of an excess of the ubiquitous competitor cysteine. In vitro viability assays showed no toxicity of <b>Re-Py-PhenISA</b> and <b>Ru-PhenISA</b> complexes, at concentrations in the range of 0.5–50 μM (calculated on the metal-containing unit), toward HEK-293 (human embryonic kidney) cells. A preliminary investigation of internalization in HEK-293 cells, by means of fluorescence confocal microscopy, showed that <b>Ru-PhenISA</b> enters cells via an endocytic pathway and, subsequently, homogeneously diffuse within the cytoplasm across the vesicle membranes

Thermal stability of Met-G-CSF and of the two pegylated forms by CD and fluorescence spectroscopies.

<p>A) Temperature dependence of the CD intensity at 222 nm. B) Temperature dependence of the HT[V] intensity at 222 nm. C) Temperature dependence of the fluorescence emission at 330 nm (excitation at 295 nm). D) Temperature dependence of the ratio between the fluorescence emission at 330 nm and at 350 nm (excitation at 295 nm).</p

FTIR spectroscopy analysis of Met-G-CSF and of the two pegylated forms.

<p>A) FTIR spectra of non-pegylated Met-G-CSF and of the two isomeric Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG. B) Second derivative spectra in the Amide I band of the three proteins as in A).</p

Dynamic light scattering and second derivative FTIR spectra of non-pegylated Met-G-CSF and of the two isomeric Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG.

<p>A) Light scattering intensity, in arbitrary units, as a function of the incubation time at 55°C. At the zero time the bath temperature was changed from 37°C to 55°C. B) Polydispersity index (Eq.3) of the non-pegylated Met-G-CSF and of the two isomeric pegylated proteins as a function of the incubation time. C) Distribution of the hydrodynamic radii corresponding to the most abundant component in the DLS decay. The solid and dashed lines are best fit Gaussian function to the data that corresponds to Rh = 39.4±4 nm and Rh = 70±20 nm for the pegylated and non-pegylated samples respectively. D) Met-G-CSF, Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG second derivative FTIR spectra measured after 7 hours of incubation at 55°C.Concerning the size of the protein aggregates, the diffusion coefficients (Eq.1) were used to evaluate the protein aggregate hydrodynamic radii through Eq.2. Non-pegylated Met-G-CSF displayed an average hydrodynamic radius of 1.4±0.4 nm at 37°C with negligible presence of protein aggregates. At 55°C, after thermal equilibrium was reached, we found instead a prevalent component with R_h = 70±20 nm. It should be noted that the value of 70 nm represents the average size of protein aggregates still in solution after 7 hours of incubation at 55^oC.</p

Thermal stability of Met-G-CSF and of the two pegylated forms by CD spectroscopy.

<p>CD spectra of non-pegylated Met-G-CSF and of the two isomeric Met-G-CSF-Met1-PEG and Met-G-CSF-Gln135-PEG at 20°C (continuous line), 95°C (dashed line), and 20°C after heating up to 95°C (dotted line).</p

Thermal and Chemical Stability of Thiol Bonding on Gold Nanostars

The stability of thiol bonding on the surface of star-shaped gold nanoparticles was studied as a function of temperature in water and in a set of biologically relevant conditions. The stability was evaluated by monitoring the release of a model fluorescent dye, Bodipy-thiol (BDP-SH), from gold nanostars (GNSs) cocoated with poly­(ethylene glycol) thiol (PEG-SH). The increase in the BDP-SH fluorescence emission, quenched when bound to the GNSs, was exploited to this purpose. A maximum 15% dye release in aqueous solution was found when the bulk temperature of gold nanostars solutions was increased to <i>T</i> = 42 °C, the maximum physiological temperature. This fraction reduces 3–5% for temperatures lower than 40 °C. Similar results were found when the temperature increase was obtained by laser excitation of the near-infrared (NIR) localized surface plasmon resonance of the GNSs, which are photothermally responsive. Besides the direct impact of temperature, an increased BDP-SH release was observed upon changing the chemical composition of the solvent from pure water to phosphate-buffered saline and culture media solutions. Moreover, also a significant fraction of PEG-SH was released from the GNS surface due to the increase in temperature. We monitored it with a different approach, that is, by using a coating of α-mercapto-ω-amino PEG labeled with tetramethylrhodamine isothiocyanate on the amino group, that after heating was separated from GNS by ultracentrifugation and the released PEG was determined by spectrofluorimetric techniques on the supernatant solution. These results suggest some specific limitations in the use of the gold–thiolate bond for coating of nanomaterials with organic compounds in biological environments. These limitations come from the duration and the intensity of the thermal treatment and from the medium composition and could also be exploited in biological media to modulate the in vivo release of drugs

A Molecular Thermometer for Nanoparticles for Optical Hyperthermia

We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within ≅20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/°C) and low uncertainty (±0.3 °C). Gold nanostars are ≅3 and ≅100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia