Improved Stability of “Naked” Gold Nanoparticles Enabled by in Situ Coating with Mono and Multivalent Thiol PEG Ligands

Unprotected (“naked”) gold nanoparticles with high monodispersity (⟨<i>d</i>⟩, 5.5± 0.5 nm) were obtained in a facile and single-step microwave-assisted hydrolytic decomposition of the molecular precursor [NMe₄]­[Au­(CF₃)₂]. Given their chloride-free surface chemistry, the as-obtained gold nanoparticles were in situ functionalized with mono-, di-, and trivalent thiolated PEG ligands in order to study the influence of multivalent character of the ligands on the stability of the colloidal solutions. For this purpose, a novel tridentate ligand was synthesized and the previously reported syntheses of mono- and divalent thiol ligands were improved. Owing to the pristine character of the Au nanoparticles no ligand exchange was required, and the colloidal and chemical stability of the mono- and multivalent functionalized particles purely depended on the ligating ability of the thiolated groups. In situ-functionalized Au nanoparticles showed a strikingly (2 orders of magnitude higher) improved stability against small nucleophiles such as sodium cyanide compared to gold nanoparticles coated with citrate ligands and functionalized via a ligand-exchange reaction. The monovalent thiol PEG ligand produced most stable colloids against cyanide, which is explained by a strongly increased numerical ligand-density on the surface. Gold colloids stabilized by di- and trivalent ligands exhibited high stability in aqueous solutions with high NaCl concentrations (2 M) in contrast to those functionalized with the monovalent PEG ligand, which were only temporally stable in dilute NaCl solutions. The beneficial effect of the multivalence of the ligands was further demonstrated by the incorporation of an additional chelating ligand (dithiothreitol) to the colloidal dispersions

Single-cell RT-PCR following Ca²⁺ imaging and cell isolation.

<p>(<b>A</b>) Representative intracellular Ca²⁺ increase (F340/380 ratio, arbitrary units) of a VSN (cell “A”) loaded with fura-2 in response to the sulfated steroid E1050 (chemical structure shown on the side), but not to urine high molecular weight fraction (HMW). (B) Ratiometric (340/380) imaging of the cell shown in A during stimulation with control buffer (DMSO) and E1050. Responsive cells are later picked using a glass capillary micropipette. Arrowhead points to the cell before and after (inside of the micropipette) picking. Scale bar, 10 μm. (C) Ethidium-bromide stained agarose gels of RT-PCR products generated from 5 cells (A to E) showing Ca²⁺ responses to E1050, a single cell lacking responses (n/r), and two water controls (w1 and w2). cDNA collected from pooled whole VNOs was used as positive control (VNO). PCR amplification of cDNA collected from single cells was performed using gene specific primers for <i>Omp</i>, <i>Gnao1</i> (Gαo), <i>Gnai2</i> (Gαi2) and degenerate primers for three members of the <i>V1rj</i> family.</p

Functional overexpression of <i>V1rj2</i> in HSV-infected VSNs.

<p>(A) Diagram of the HSV-1 expression cassettes. In control amplicons <i>GFP</i> is expressed under the HSV-1 IE4/5 promoter, whereas in V1rj2 amplicons <i>V1rj2</i> cDNA is inserted in the multi cloning site (MCS) upstream of IRES and GFP. (B) Bright field (BF), GFP and pseudocolor fura-2 images of a representative VSN infected with HSV-<i>V1rj2</i>-IRES-<i>GFP</i> and activated by the mix of four sulfated estrogens (E mix). Time course of intracellular calcium is shown on the right. Stimulations were 30 s long. Scale bar, 10 μm. (C) Color-coded heat map of normalized Δratio responses (Δratio_norm) from 26 GFP+ VSNs infected with <i>V1rj2</i>-IRES-<i>GFP</i> which showed responses to the estrogen mix (E mix). These cells lacked responses to other stimuli except for high K⁺. (D) Comparison of response amplitudes expressed as normalized Δratio responses to E mix and high K of 26 individual cells shown in C. Each neuron is shown as a separate circle. Red line represents linear fit (slope = 0.55). Responses to E mix and high K⁺ are not significantly different (p = 0.92, Mann-Whitney test). (E) Summary of GFP+ cell activation for every stimulus on VSNs infected either with V1rj2-GFP (green) or with GFP control amplicons (grey). Values are expressed as percentage (%) of activated cells from the total of GFP+ cells. VSNs expressing <i>V1rj2-GFP</i> show increased cell responsivity to E mix (p < 0.005) but not to E2734, HMW, DMSO or high K⁺ (p = 0.1–0.4, Student’s t test). Responses to DMSO, E2734 or HMW did not overlap with responses to E-mix. N = 436 <i>V1rj2-GFP</i> cells and 563 control-<i>GFP</i> cells, in 8 and 16 experiments, respectively.</p

Herpes simplex virus type 1 (HSV-1) expression system in HEK cells and VSNs.

<p>(A) HEK cells transfected with pHSV-<i>V1rj2</i>-IRES-<i>GFP</i> expression vector do not show responses to a mix of sulfated steroids (E mix: E1100, E0893, E0588, and E1050, each 100 μM), HMW nor E2734 in Ca²⁺ imaging. (B) Infection of HEK cells (top panels) and freshly prepared VSNs (bottom panels) with HSV-GFP amplicon virus monitored at three different time points (6 h, 24 h and 48 h). Scale bars, 50 μm (C) Left and center, measures of fluorescence intensity (in arbitrary units, a.u.) on infected single HEK cells and VSNs. Right, normalized abundance of infected VSNs (GFP+) at each time point. (D) Single VSNs infected with HSV-GFP virus for 20 h and loaded with fura-2. Bright field (BF), GFP and F340/380 ratio images of an infected cell are shown. Average rate of infection was 23% (N = 16 402 cells in 69 infections). Infection rate for specific batches of HSV: GFP, 19% (N = 10 171); V1rj2, 15% (N = 3109); V2r1b, 27% (N = 2675); Fpr3, 39% (N = 447). (E) VSNs prepared from OMP-GFP mice infected with a HSV-mCherry virus. Of all mCherry-positive cells 76% were also positive for GFP. N = 978 mCherry+ cells in 14 infections. Scale bars, 10 μm.</p

Viral transduction of <i>V2r1b</i> and <i>Fpr3</i> receptors in VSNs.

<p>(A) Bright field (BF), GFP and pseudocolor fura-2 images of a VSN infected with HSV-<i>V2r1b</i>-IRES-<i>GFP</i> (arrowhead) and activated by the MHC binding peptide SYFPEITHI (SYF). The neighboring non-infected GFP-negative cell (arrow) does not show any calcium increase during peptide stimulation. (B) Summary of cell responses to different stimuli. A 6-fold increase of responsivity to SYF is observed in <i>V2r1b-GFP</i> cells (p < 0.005, Student t test), but not to HMW fraction, the mitochondria-derived peptide ND1, or high K⁺. (C) Enhanced responsivity to SYF is not observed in Gαo-deficient mice (cGαo^-/-) VSNs infected with HSV-<i>V2r1b</i>-IRES-<i>GFP</i>. (D) A single HSV-<i>Fpr3</i>-IRES-<i>GFP</i> infected VSN activated by the synthetic hexapeptide W-peptide (w-pep) is shown. (E) <i>Fpr3-GFP</i> cells show a significantly enhanced number of responses to W-peptide versus GFP control cells (p < 0.001, Student t test), but not to HMW, ND1 or high K⁺. N = 469 <i>V2r1b-GFP</i>, 163 <i>Fpr3-GFP</i> and 1224 control-GFP cells, in 12, 8 and 27 experiments, respectively. Scale bars, 10 μm.</p

Electronic and Exchange Coupling in a Cross-Conjugated D–B–A Biradical: Mechanistic Implications for Quantum Interference Effects

A combination of variable-temperature EPR spectroscopy, electronic absorption spectroscopy, and magnetic susceptibility measurements have been performed on Tp^Cum,MeZn­(SQ-<i>m-</i>Ph-NN) (<b>1-meta</b>) a donor–bridge–acceptor (D–B–A) biradical that possesses a cross-conjugated <i>meta</i>-phenylene (<i>m-</i>Ph) bridge and a spin singlet ground state. The experimental results have been interpreted in the context of detailed bonding and excited-state computations in order to understand the excited-state electronic structure of <b>1-meta</b>. The results reveal important excited-state contributions to the ground-state singlet–triplet splitting in this cross-conjugated D–B–A biradical that contribute to our understanding of electronic coupling in cross-conjugated molecules and specifically to quantum interference effects. In contrast to the conjugated isomer, which is a D–B–A biradical possessing a <i>para</i>-phenylene bridge, admixture of a single low-lying singly excited D → A type configuration into the cross-conjugated D–B–A biradical ground state makes a negligible contribution to the ground-state magnetic exchange interaction. Instead, an excited state formed by a Ph-NN (HOMO) → Ph-NN (LUMO) one-electron promotion configurationally mixes into the ground state of the <i>m-</i>Ph bridged D–A biradical. This results in a double (dynamic) spin polarization mechanism as the dominant contributor to ground-state antiferromagnetic exchange coupling between the SQ and NN spins. Thus, the dominant exchange mechanism is one that activates the bridge moiety via the spin polarization of a doubly occupied orbital with phenylene bridge character. This mechanism is important, as it enhances the electronic and magnetic communication in cross-conjugated D–B–A molecules where, in the case of <b>1-meta</b>, the magnetic exchange in the active electron approximation is expected to be <i>J</i> ∼ 0 cm^–1. We hypothesize that similar superexchange mechanisms are common to all cross-conjugated D–B–A triads. Our results are compared to quantum interference effects on electron transfer/transport when cross-conjugated molecules are employed as the bridge or molecular wire component and suggest a mechanism by which electronic coupling (and therefore electron transfer/transport) can be modulated

Revisiting the Polyoxometalate-Based Late-Transition-Metal-Oxo Complexes: The “Oxo Wall” Stands

Terminal oxo complexes of the late transition metals Pt, Pd, and Au have been reported by us in <i>Science</i> and <i>Journal of the American Chemical Society</i>. Despite thoroughness in characterizing these complexes (multiple independent structural methods and up to 17 analytical methods in one case), we have continued to study these structures. Initial work on these systems was motivated by structural data from X-ray crystallography and neutron diffraction and ¹⁷O and ³¹P NMR signatures which all indicated differences from all previously published compounds. With significant new data, we now revisit these studies. New X-ray crystal structures of previously reported complexes K₁₄[P₂W₁₉O₆₉(OH₂)] and “K₁₀Na₃[Pd^IV(O)­(OH)­WO­(OH₂)­(PW₉O₃₄)₂]” and a closer examination of these structures are provided. Also presented are the ¹⁷O NMR spectrum of an ¹⁷O-enriched sample of [PW₁₁O₃₉]^7– and a careful combined ³¹P NMR-titration study of the previously reported “K₇H₂[Au­(O)­(OH₂)­P₂W₂₀O₇₀(OH₂)₂].” These and considerable other data collectively indicate that previously assigned terminal Pt-oxo and Au-oxo complexes are in fact cocrystals of the all-tungsten structural analogues with noble metal cations, while the Pd-oxo complex is a disordered Pd­(II)-substituted polyoxometalate. The neutron diffraction data have been re-analyzed, and new refinements are fully consistent with the all-tungsten formulations of the Pt-oxo and Au-oxo polyoxometalate species