Multicopper Clusters Enable Oxidative Phenol Macrocyclization (OxPM) of Peptides.

The biosynthesis of glycopeptide antibiotics such as vancomycin and other biologically active biaryl-bridged and diaryl ether-linked macrocyclic peptides includes key enzymatic oxidative phenol macrocyclization(s) of linear precursors. However, a simple and step-economical biomimetic version of this transformation remains underdeveloped. Here, we report highly efficient conditions for preparing biaryl-bridged and diaryl ether-linked macrocyclic peptides based on multicopper(II) clusters. The selective syntheses of ring models of vancomycin and the arylomycin cyclic core illustrate the potential of this technology to facilitate the assembly of complex antibiotic macrocyclic peptides, whose syntheses are considered highly challenging. The unprecedented ability of multicopper(II) clusters to chelate tethered diphenols and promote intramolecular over intermolecular coupling reactions demonstrates that copper clusters can catalyze redox transformations that cannot be accessed by smaller metal catalysts.

PVP-coated Gd-grafted nanodiamonds as a novel and potentially safer contrast agent for in vivo MRI.

PURPOSE: Testing the potential use of saline suspension of polyvinylpyrrolidone (PVP)-coated gadolinium(Gd)-grafted detonation nanodiamonds (DND) as a novel contrast agent in MRI. METHODS: Stable saline suspensions of highly purified de-agglomerated Gd-grafted DND particles coated by a PVP protective shell were prepared. T1 and T2 proton relaxivities of the suspensions with varying gadolinium concentration were measured at 8 Tesla. A series of ex vivo (phantom) and in vivo dynamic scans were obtained in 3 Tesla MRI using PVP-coated Gd-grafted DND and gadoterate meglumin in equal concentrations of gadolinium, and then T1 -weighted hyperintensity was compared. RESULTS: The proton relaxivities of PVP-coated Gd-grafted DND were found to be r1 = 15.9 ± 0.8 s-1 mM-1 and r2 = 262 ± 15 s-1 mM-1 , respectively, which are somewhat less than those for uncoated Gd-grafted DND but still high enough. Ex vivo MRI evaluation of PVP-coated Gd-grafted DND results with a dose-dependent T1 -weighted hyperintensity with a significant advantage over the same for gadoterate meglumin. The same was found when the 2 contrast agents were tested in vivo. CONCLUSION: The novel MRI contrast agent - saline suspensions of PVP-coated Gd-grafted DND - provides significantly higher signal intensities than the common tracer gadoterate meglumin, therefore increasing its potential for a safer use in clinics.

Mitochondria membrane transformations in colon and prostate cancer and their biological implications.

Mitochondria have emerged as important determinants in cancer progression and malignancy. However, the role of mitochondrial membranes in cancer onset and progression has not been thoroughly investigated. This study compares the structural and functional properties of mitochondrial membranes in prostate and colon cancer cells in comparison to normal mitochondria, and possible therapeutic implications of these membrane changes. Specifically, isolation of cell mitochondria and preparation of inverted sub-mitochondrial particles (SMPs) illuminated significant cancer-induced modulations of membrane lipid compositions, fluidity, and activity of cytochrome c oxidase, one of the key mitochondrial enzymes. The experimental data further show that cancer-associated membrane transformations may account for mitochondria targeting by betulinic acid and resveratrol, known anti-cancer molecules. Overall, this study probes the relationship between cancer and mitochondrial membrane transformations, underlying a potential therapeutic significance for mitochondrial membrane targeting in cancer.

Structural Characterization of Reconstituted Bioactive-Loaded Nanodomains after Embedding in Films Using Electron Paramagnetic Resonance and Self-Diffusion Nuclear Magnetic Resonance Techniques.

Pharmaceutical applications of microemulsions (MEs) as drug delivery vehicles are recently gaining scientific and practical interests. Most MEs are able to solubilize bioactive molecules, but, at present, they cannot guarantee either controlled release of the drugs or significant advantage in the bioavailability of the bioactives. This study proposes to incorporate the modified ME structures, or nanodomains, into a natural polymeric film, to be used as a stable and capacious reservoir of drug-loaded nanodomains. These nanodomain-loaded films may release the nanodroplets along with the drug molecules in a slow and controlled way. Gellan gum, an anionic polysaccharide, was used in aqueous solution as the film former, and curcumin, hydrophobic polyphenol, served as the guest molecule in the loaded systems. Films were prepared by using empty and curcumin-loaded MEs. It is imperative to verify the persistence of the ME structure upon the dissolution of the film mimicking its behavior when in contact with a human physiological aqueous environment via reaching the cell membranes. For this purpose, the films were dissolved, and the reconstituted ME structure was compared with the ME structure before film formation. Characterization of these structures, before and after dissolution, was achieved using electron paramagnetic resonance (EPR) and self-diffusion nuclear magnetic resonance (SD-NMR) techniques. Specific spin probes were inserted in the system, and a computer-aided analysis of the EPR spectra was performed to provide information on nanodomain microstructure assemblies. In addition, the SD-NMR profile of each component was analyzed to extract information on the diffusivity of the ME components before film formation and after ME reconstitution. The EPR and SD-NMR results were in good agreement to each other. The most important finding was that, after film dissolution, the ME nanodomains were reversibly and spontaneously reformed. It was also found that the film did not perturb the ME-nanodomain structure embedded in it. The film remained transparent and the bioactive curcumin was easily solubilized into the ME-droplet/water interface even after film dissolution. The combined techniques confirmed that the film constituted by bioactive-loaded MEs can serve as novel drug delivery vehicles.

Behavior of PPI-G2 Dendrimer in a Microemulsion.

Dendrimer nanostructures are of eminent interest in biomedical applications because of their uniform and well-defined molecular size and shape, and their ability to cross cell membranes and reduce the risk of premature clearance from the human body. Dendrimers perform as gene and drug carriers and have also shown significant therapeutic properties for treating cancer and neurodegenerative diseases. A complex drug delivery system, based on a dendrimer solubilized in the aqueous core of a water-in-oil (W/O) microemulsion (ME) along with the drug may combine the advantages of both dendrimers and MEs to provide better control of drug release. We propose a new microemulsion composed of drug-permitted surfactants and dendrimer that can be used as a potential controlled drug delivery nanosystem. The influence of second generation poly(propyleneimine) (PPI-G2) dendrimer; solubilized in (W/O) ME with a capacity of up to 25 wt% PPI-G2 at various pHs; and their interactions with the surfactant phosphatidylcholine (PC), cosurfactant (butanol), and water was studied. SAXS and EPR measurements indicated that increasing PPI-G2 concentration reduces droplet curvature and increases droplet size thus increasing macro-(SAXS) and micro-(EPR) order degree. Furthermore, SD-NMR and ATR-FTIR show stronger interactions between PPI-G2 and water molecules at the expense of PC and butanol headgroups hydration, which increases microviscosity (EPR). PPI-G2's effect is somewhat opposite to the increasing water phase effect, thus reducing the amount of free water (DSC) and slowing the mobility of all ME components (SD-NMR).

Covalent binding of a nickel macrocyclic complex to a silica support: towards an electron exchange column.

Ni(II)L(2), L(2) = 1-propyl-1,3,5,8,12-pentaazacyclotetradecane, was covalently bound to a silica support. This complex can be reversibly oxidized to the corresponding Ni(III) complex. The latter complex is relatively long lived. Therefore electron exchange columns based on this material can be prepared.

Bolaamphiphilic vesicles encapsulating iron oxide nanoparticles: new vehicles for magnetically targeted drug delivery.

Bolaamphiphiles - amphiphilic molecules consisting of two hydrophilic headgroups linked by a hydrophobic chain - form highly stable vesicles consisting of a monolayer membrane that can be used as vehicles to deliver drugs across biological membranes, particularly the blood-brain barrier (BBB). We prepared new vesicles comprising bolaamphiphiles (bolavesicles) that encapsulate iron oxide nanoparticles (IONPs) and investigated their suitability for targeted drug delivery. Bolavesicles displaying different headgroups were studied, and the effect of IONP encapsulation upon membrane interactions and cell uptake were examined. Experiments revealed more pronounced membrane interactions of the bolavesicles assembled with IONPs. Furthermore, enhanced internalization and stability of the IONP-bolavesicles were observed in b.End3 brain microvessel endothelial cells - an in vitro model of the blood-brain barrier. Our findings indicate that embedded IONPs modulate bolavesicles' physicochemical properties, endow higher vesicle stability, and enhance their membrane permeability and cellular uptake. IONP-bolavesicles thus constitute a promising drug delivery platform, potentially targeted to the desired location using external magnetic field.

W/O microemulsions as dendrimer nanocarriers: an EPR study.

A complex system, based on a dendrimer solubilized in the aqueous core of water-in-oil microemulsion, may combine the advantages of both dendrimers and microemulsions to provide better control of drug release. We report for the first time the use of EPR technique to determine the effect of solubilized dendrimer on the structure of the microemulsion. The solubilized poly(propyleneimine) (PPI-G2) interacts with sodium bis(2-ethylhexyl) sulfosuccinate (AOT). EPR analysis provided information on polarity, microviscosity, and molecular order of the systems. Polarity and microviscosity increased from unloaded water-in-oil microemulsion to the system loaded with 0.2 wt % PPI-G2, but remained unchanged with higher PPI-G2 loads. The degree of order also increased with 0.2 wt % PPI-G2 with only minor additional increase with larger quantities (25 wt %) of PPI-G2. Variations in pH only slightly affected the structure of microemulsion in the absence and presence of the loaded dendrimers. Aliphatic oils with longer lipophilic chains enhanced the structural order of the microemulsion. On increasing water content, polarity and degree of order increased. PPI-G2 dendrimer in small loads is attracted by the negatively charged AOT and thus intercalates in the interface of the droplets. Yet, at higher PPI-G2 loads, the excess molecules are solubilized in the water core.

Detection of nitric oxide from pig trachea by a fluorescence method.

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO(2). Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2 nmol/g x min, and NO-synthase stimulated by extracellular adenosine 5'-triphosphate produced NO flux of 0.9 nmol/g x min.

Heterogeneity in the structural dynamics of Sulfolobus solfataricus beta-glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry.

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.