[show abstract][hide abstract] ABSTRACT: The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and because ferrous iron, but not ferric iron, is carried by the divalent metal transporter DMT-1. Moreover, ferrous iron frequently causes gastrointestinal side effects. Iron hydroxide nanoparticles with neutral and hydrophilic carbohydrate shells are alternatively used to ferrous salts. In these formulations gastrointestinal side effects are rare because hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by the solubilizing shell; nevertheless, iron bioavailability is even worse compared to ferrous salts. In this study the cell uptake of iron hydroxide and iron oxide nanoparticles (FeONP) with negatively charged shells of different chemical types and sizes was compared to the uptake of those with neutral hydrophilic shells, ferrous sulfate and ferric chloride. The nanoparticle uptake was measured in Caco-2 cells with the iron detecting ferrozine method and visualized by transmission electron microscopy. The toxicity was evaluated using the MTT assay. For nanoparticles with a negatively charged shell the iron uptake was about 40 times higher compared to those with neutral hydrophilic carbohydrate shell or ferric chloride and in the same range as ferrous sulfate. However, in contrast to ferrous sulfate, nanoparticles with negatively charged shells showed no toxicity. Two different uptake mechanisms were proposed: diffusion for hydroxide nanoparticles with neutral hydrophilic shell and adsorptive endocytosis for nanoparticles with negatively charged shells. It needs to be determined whether iron hydroxide nanoparticles with negatively charged shells also show improved bioavailability in iron-deficient patients compared to iron hydroxide nanoparticles with a neutral hydrophilic shell, which exist in the market today.
[show abstract][hide abstract] ABSTRACT: For studying proliferation and determination of survival of cancer cells after irradiation, the multiple MTT assay, based on the reduction of a yellow water soluble tetrazolium salt to a purple water insoluble formazan dye by living cells was modified from a single-point towards a proliferation assay. This assay can be performed with a large number of samples in short time using multi-well-plates, assays can be performed semi-automatically with a microplate reader. Survival, the calculated parameter in this assay, is determined mathematically. Exponential growth in both control and irradiated groups was proven as the underlying basis of the applicability of the multiple MTT assay. The equivalence to a clonogenic survival assay with its disadvantages such as time consumption was proven in two setups including plating of cells before and after irradiation. Three cell lines (A 549, LN 229 and F 98) were included in the experiment to study its principal and general applicability.
[show abstract][hide abstract] ABSTRACT: The flow of bile secretion into the human digestive system was simulated by the dilution of a bile salt-lipid micellar solution. The structural development upon the dilution of the fed state bile model FeSSIF(mod6.5) to the fasted state bile model FaSSIF(mod) was investigated by small-angle neutron scattering (SANS) and dynamic light scattering (DLS) in crossed beam experiments to observe small and large structures in a size range of 1 nm to 50 μm in parallel. Because of the physiologically low lipid and surfactant concentrations of 2.625 mM egg-phosphatidylcholine and 10.5 mM taurocholate the sensitivity of the neutron-structural investigations was improved by partial solvent deuteration with 71% D(2)O, with control experiments in H(2)O. Static experiments of initial and end state systems after 6 days of development revealed the presence of mixed bile salt-lipid micelles of 5.1 nm size in the initial state model FeSSIF(mod6.5), and large liposomes in FaSSIF(mod), which represent the late status after dilution of bile secretion in the intestine in the fasted state. The liposomes depicted a size of 34.39 nm with a membrane thickness of 4.75 nm, which indicates medium to large size unilamellar vesicles. Crossed beam experiments with time-resolved neutron and light scattering experiments after fast mixing with a stopped-flow device revealed a stepwise structural dynamics upon dilution by a factor of 3.5. The liposome formation was almost complete five minutes after bile dilution. The liposomes 30 min after dilution resembled the liposomes found after 6 days and depicted a size of 44.56 nm. In the time regime between 3 and 100 s a kinetic intermediate was observed. In a further experiment the liposome formation was abolished when the dilution was conducted with a surfactant solution containing sodium dodecyl sulfate.
[show abstract][hide abstract] ABSTRACT: The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and ferrous iron but not ferric iron is carried by the divalent metal transporter DMT-1. Moreover, ferrous iron frequently causes gastrointestinal side effects. In iron(III)-hydroxide nanoparticles hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by a solubilising carbohydrate shell, yet bioavailability from such particles is insufficient when compared with ferrous salts. To increase their intestinal uptake iron(III)-hydroxide nanoparticles were coupled in this study with the protoporphyrin hemin, which undergoes carrier-mediated uptake in the intestine.
Uptake of iron(III)-hydroxide nanoparticles with hemin covalently coupled by DCC reaction was measured in Caco-2 cells with a colorimetric assay and visualized by transmission electron microscopy.
Nanoparticles were taken up by carrier-mediated transport, since uptake was temperature-dependent and increased with an increasing hemin substitution grade. Furthermore, uptake decreased with an increasing concentration of free hemin, due to competition for carrier-mediated uptake.
Hemin-coupled iron(III)-hydroxide nanoparticles were carried by a heme specific transport system, probably via receptor mediated endocytosis. It can be expected that this system shows improved absorption of iron compared with uncoupled iron(III)-hydroxide nanoparticles, which exist on the market today.
The Journal of pharmacy and pharmacology. 12/2011; 63(12):1522-30.
[show abstract][hide abstract] ABSTRACT: In biological fluids, proteins associate with nanoparticles, leading to a protein "corona" defining the biological identity of the particle. However, a comprehensive knowledge of particle-guided protein fingerprints and their dependence on nanomaterial properties is incomplete. We studied the long-lived ("hard") blood plasma derived corona on monodispersed amorphous silica nanoparticles differing in size (20, 30, and 100 nm). Employing label-free liquid chromatography mass spectrometry, one- and two-dimensional gel electrophoresis, and immunoblotting the composition of the protein corona was analyzed not only qualitatively but also quantitatively. Detected proteins were bioinformatically classified according to their physicochemical and biological properties. Binding of the 125 identified proteins did not simply reflect their relative abundance in the plasma but revealed an enrichment of specific lipoproteins as well as proteins involved in coagulation and the complement pathway. In contrast, immunoglobulins and acute phase response proteins displayed a lower affinity for the particles. Protein decoration of the negatively charged particles did not correlate with protein size or charge, demonstrating that electrostatic effects alone are not the major driving force regulating the nanoparticle-protein interaction. Remarkably, even differences in particle size of only 10 nm significantly determined the nanoparticle corona, although no clear correlation with particle surface volume, protein size, or charge was evident. Particle size quantitatively influenced the particle's decoration with 37% of all identified proteins, including (patho)biologically relevant candidates. We demonstrate the complexity of the plasma corona and its still unresolved physicochemical regulation, which need to be considered in nanobioscience in the future.
[show abstract][hide abstract] ABSTRACT: The treatment of iron deficiency anemia with polynuclear iron formulations is an established therapy in patients with chronic kidney disease but also in other disease areas like gastroenterology, cardiology, oncology, pre/post operatively and obstetrics' and gynecology. Parenteral iron formulations represent colloidal systems in the lower nanometer size range which have traditionally been shown to consist of an iron core surrounded by a carbohydrate shell. In this publication, we for the first time describe the novel matrix structure of iron isomaltoside 1000 which differs from the traditional picture of an iron core surrounded by a carbohydrate. Despite some structural similarities between the different iron formulations, the products differ significantly in their physicochemical properties such as particle size, zeta potential, free and labile iron content, and release of iron in serum. This study compares the physiochemical properties of iron isomaltoside 1000 (Monofer) with the currently available intravenous iron preparations and relates them to their biopharmaceutical properties and their approved clinical applications. The investigated products encompass low molecular weight iron dextran (CosmoFer), sodium ferric gluconate (Ferrlecit), iron sucrose (Venofer), iron carboxymaltose (Ferinject/Injectafer), and ferumoxytol (Feraheme) which are compared to iron isomaltoside 1000 (Monofer). It is shown that significant and clinically relevant differences exist between sodium ferric gluconate and iron sucrose as labile iron formulations and iron dextran, iron carboxymaltose, ferumoxytol, and iron isomaltoside 1000 as stable polynuclear formulations. The differences exist in terms of their immunogenic potential, safety, and convenience of use, the latter being expressed by the opportunity for high single-dose administration and short infusion times. Monofer is a new parenteral iron product with a very low immunogenic potential and a very low content of labile and free iron. This enables Monofer, as the only IV iron formulation, to be administered as a rapid high dose infusion in doses exceeding 1000 mg without the application of a test dose. This offers considerable dose flexibility, including the possibility of providing full iron repletion in a single infusion (one-dose iron repletion).
European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 03/2011; 78(3):480-91. · 3.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: We describe the synthesis of linear-hyperbranched lipids for liposome preparation based on linear poly(ethylene glycol) (PEG) and hyperbranched polyglycerol (PG). Molecular weights were adjusted to values around 3000 g/mol with varying degrees of polymerization of the linear and the branched segments in analogy to PEG-based stealth lipids; polydispersities were generally low and below 1.3. The hydrophobic anchors were introduced into the lipid structures as initiators for the anionic polymerization of ethylene oxide and are either based on cholesterol or on different aliphatic glyceryl ethers. Complete incorporation of the apolar initiators was evidenced by MALDI-ToF analysis at all stages of the reaction. The linear-hyperbranched polyether lipid is incorporated as the polyfunctional shell in liposome formulations together with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The resulting liposomes were subsequently characterized via dynamic light scattering (DLS) and small angle neutron scattering (SANS) as well as transmission electron microscopy (TEM), demonstrating the formation of unilamellar liposomes in the size range of 40 to 50 nm.
[show abstract][hide abstract] ABSTRACT: We report the vectorial incorporation of a highly asymmetric F0F1 ATP synthase complex from Micrococcus luteus into polymer-supported membranes. Dynamic light scattering and cryo electron microscopy confirm that the use of weak surfactants (bile acid) allows for the non-disruptive protein incorporation into lipid vesicles. Spreading of vesicles with ATP synthase onto a cellulose support results in a homogeneous distribution of proteins, in contrast to a patchy image observed on bare glass slides. The orientation of ATP synthase can be identified using an antibody to the ATP binding site as well as from topographic profiles of the surface. The method to "align" transmembrane proteins in supported membranes would open a possibility to quantify protein functions in biomimetic model systems.
[show abstract][hide abstract] ABSTRACT: The aim of these studies was to compare dialysis and dispersion methods for determining in vitro release of propranolol, metoprolol, pindolol, and atenolol from multilamellar liposomes. Multilamellar vesicles (MLV) were prepared using hydrogenated soy-lecithin phospholipon 90H (Ph 90H) as the primary lipid. The same volume of pH 7.4 phosphate buffered saline was used as a receptor medium for both methods. Samples were withdrawn, and drug concentration was determined using HPLC. All drug-containing liposomes exhibited an initial burst release followed by a slower rate of release. The rate and extent of drug release from MLV was dependent on the physicochemical properties of the drug. For all drugs investigated, the rate of release was higher for the dispersion method as compared with the dialysis method.
[show abstract][hide abstract] ABSTRACT: A cytochrome aa3 terminal oxidase was isolated from protoplast membrane vesicles of Micrococcus luteus grown under aerobic conditions. The purified complex showed similarities to cytochrome c oxidase (EC 22.214.171.124) of the electron transport chain of mitochondria and many prokaryotes. The enzyme was solubilized by subsequent treatment with the detergents CHAPS and and purified by ion-exchange chromatography using poly-l-lysine agarose and TMAE-fractogel-650 (S) columns, followed by hydroxyapatite chromatography. The purified complex is composed of two major subunits with apparent molecular masses of 54 and 32 kDa. After purification the isolated enzyme contains 12.1 nmol of heme A (mg protein)−1 and exhibits absorption maxima at 424 nm and 598 nm in the oxidized state and at 442 nm and 599 nm in the reduced state. The CO-difference spectrum shows peaks at 428 and 590 nm which is indicative of heme a3, furthermore oxygen consumption was found to be sensitive to cyanide.
Fems Microbiology Letters - FEMS MICROBIOL LETT. 01/1994; 124(2):173-178.
[show abstract][hide abstract] ABSTRACT: Carbodiimide-mediated peptide synthesis in aqueous solution has been studied with respect to self-ordering of amino acids. The copolymerisation of amino acids in the presence of glutamic acid or pyroglutamic acid leads to short pyroglutamyl peptides. Without pyroglutamic acid the formation of higher polymers is favoured.The interactions of the amino acids and the peptides, however, are very complex. Therefore, the experimental results are rather difficult to explain. Some of the experimental results, however, can be explained with the aid of computer simulation programs. Regarding only the tripeptide fraction the copolymerisation of pyroGlu, Ala and Leu, as well as the simulated copolymerisation lead to pyroGlu-Ala-Leu as the main reaction product. The amino acid composition of the insoluble peptides formed during the copolymerisation of Ser, Gly, Ala, Val, Phe, Leu and Ile corresponds in part to the computer-simulated copolymerisation data.
Origins of Life and Evolution of Biospheres 11/1984; 14(1):213-220. · 1.83 Impact Factor