Effect of fibronectin on the binding of antithrombin III to immobilized heparin

An objective of this research is to verify the mechanism of anticoagulant activity of surface-immobilized heparin in the presence of plasma proteins. The competition and binding interaction between immobilized heparin and antithrombin III (ATIII)/thrombin have been described in vitro. However, the strong ionic character of heparin leads to its specific and nonspecific binding with many other plasma proteins. Most notably, fibronectin contains six active binding sites for heparin which may interfere with the subsequent binding of heparin with ATIII or thrombin. \ud Heparin was covalently immobilized through polyethylene oxide (PEO) hydrophilic spacer groups onto a model surface synthesized by random copolymerization of styrene and p-aminostyrene. The binding interaction of immobilized heparin with ATIII was then determined in the presence of different fibronectin concentrations. The binding interaction was studied by first binding immobilized heparin with ATIII, followed by the introduction of fibronectin; heparin binding with fibronectin, followed by incubation with ATIII, and simultaneous incubation of surface immobilized heparin with ATIII and fibronectin. The extent of ATIII binding to heparin in each experiment was assayed using a chromogenic substrate for ATIII, S-2238. \ud The results of this study demonstrate that the displacement of ATIII from immobilized heparin was proportional to the fibronectin concentration, and was reversible. Furthermore, the binding sequence did not play a role in the final concentration of ATIII bound to immobilized heparin

Molecular separation by thermosensitive hydrogelmembranes

A new method for separation of molecules of different size is presented. The method is a useful addition to conventional separation methods which depend mainly on gel permeation chromatography using size exclusion. In the new method, hydrogel membranes are used which swelling level can be thermally controlled. In this study, a crosslinked poly(N-isopropylacrylamide¿co-butylmethacrylate 95:5mol%) membrane is used and three solutes of distinct molecular size: two dextrans with molecular weights of 150,000 and 4,400 g/mol respectively and uranine with a molecular weight of 376 g/mol. The swelling of the membranes as function of temperature was measured as well as the influence of the swelling level on the permeability of the three solutes. the influence of the swelling level and the solute size on the permeability was as expected from the free-volume theory. Based on these permeability phenomena, separation was performed in a continuous way by varying the membrane swelling at the appropriate time. A linear relationship between inverse membrane hydration and solute diffusion was found for uranine and dextran (MW=4,400), indicating the validity of the free-volume theory

Tissue compatibility of poly(hydroxypropylglutamate)-prazosin conjugates

Biocompatibility of an injectable biodegradable drug delivery system for prazosin was investigated in Sprague-Dawley rats by histological studies after subcutaneous injection ofpoly(hydroxypropyl glutamate)-prazosin (PHPG-prazosin) conjugate particles. The studies showed that (1) the acute inflammatory response to this injectable biodegradable polymeric prodrug system was mild and of only short duration, (2) the chronic inflammation was minimal to zero, (3) the fibrous capsule could be seen starting from 7 days and became more prominent at longer time periods, (4) a collagen network was formed into the injection site after 21 days, (5) the macrophages and foreign giant cells reacted to the globules of conjugate particles, and (6) no adverse reactions were identified. Focal inflammation and the formation of the fibrous capsule around the injection site were the significant histological findings in the histopathological studies. Therefore, it is concluded that the biodegradable injectable PHPG-prazosin carbamate polymeric prodrug system is tissue biocompatible

Detection of motional ground state population of a trapped ion using delayed pulses

Efficient preparation and detection of the motional state of trapped ions is important in many experiments ranging from quantum computation to precision spectroscopy. We investigate the stimulated Raman adiabatic passage (STIRAP) technique for the manipulation of motional states in a trapped ion system. The presented technique uses a Raman coupling between two hyperfine ground states in $^{25}$Mg$^+$, implemented with delayed pulses, which removes a single phonon independent of the initial motional state. We show that for a thermal state the STIRAP population transfer is more efficient than a stimulated Raman Rabi pulse on a motional sideband. In contrast to previous implementations, a large detuning of more than 200 times the natural linewidth of the transition is used. This approach renders STIRAP suitable for atoms in which resonant laser fields would populate fluorescing excited states and thus impede the STIRAP process. We use the technique to measure the wavefunction overlap of excited motional states with the motional ground state. This is an important application for photon recoil spectroscopy and other force sensing applications that utilize the high sensitivity of the motional state of trapped ions to external fields. Furthermore, a determination of the ground state population enables a simple measurement of the ion's temperature.Comment: 17 pages, 7 figure

Random and gradient based fields in discrete particle models of heterogeneous materials

Conventional finite element software and associated numerical models are usually used to analyze the mechanical behavior of structural scale concrete beams. However, they lack the ability of accounting for concrete heterogeneity or including concrete aging and deterioration effects. This paper introduces the advanced techniques to model the behavior of reinforced and prestressed concrete beams under shear loads utilizing a discrete particle model incorporating effects from creep, shrinkage and prestress loss. Five-meter long concrete beams with full reinforcement and different prestress levels were tested under shear loads. Material tests of the concrete, including unconfined compression, uniaxial tests to measure modulus of elasticity, and splitting tests, were carried out on the day of shear testing. Concrete creep and shrinkage measurements were also conducted to study their effect on prestress loss and consequently shear response. The model constructs the beam with concrete as discrete particles and the steel rebars/tendons as beam elements. The concrete behavior is defined by constitutive laws with its material properties calibrated based on standard experimental tests. The behavior of reinforced rebars and prestressing tendons follow the elasto-plastic law of typical steel with known Young’s modulus and yielding strength. The rebar beam elements and solid concrete elements interact with each other using penalty constraints. Before the shear load is applied, creep and shrinkage losses of concrete are evaluated, of which the parameters were calibrated based on the experimental measurements. The results of the predictive shear simulations for the reinforced and prestressed beams resemble those of the tested specimens including but not limited to the force-displacement curves, the failure types, and the crack patterns. These analysis-and-modeling techniques hold great significance to innovations in structures

Heparin release from thermosensitive hydrogels

Thermosensitive hydrogels (TSH) were synthesized and investigated as heparin releasing polymers for the prevention of surface induced thrombosis. TSH were synthesized with N-isopropyl acrylamide (NiPAAm) copolymerized with butyl methacrylate (BMA) (hydrophobic) or acrylic acid (AAc) (hydrophilic) comonomers. The incorporation of hydrophobic and hydrophilic comonomers strongly influences the swelling/shrinking behavior of TSH. Upon deswelling, gels containing the hydrophobic comonomer formed a skin-type layer, which acted as a rate controlling membrane, while the hydrophilic comonomer greatly increased gel swelling, relative to NiPAAm. Equilibrium swelling in isotonic PBS and deswelling kinetics of the synthesized gels were examined at various temperatures. The loading of heparin into the different gels was studied as a function of temperature, solution concentration, and gel composition. The release kinetics of heparin was found to be influenced by gel composition and loading temperature; the release correlated with the gel deswelling kinetics. In the case of Ni-PAAm/BMA gel, the release profile of heparin was affected by temperature dependent properties of the skin-type diffusional barrier formed on the gel surface

Release of proteins via ion exchange from albumin-heparin microspheres

Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg lysozyme on albumin-heparin microspheres was linear until saturation was abruptly reached,\ud \ud The adsorption isotherms of human lysozyme at low and high ionic strength were typical of adsorption isotherms of proteins on ion exchange gels. The adsorption of human lysozyme on albumin-heparin and albumin microspheres fit the Freundlich equation suggesting heterogeneous binding sites. This was consistent with the proposed multivalent, electrostatic interactions between human lysozyme and negatively charged microspheres. Scatchard plots of the adsorption processes of human lysozyme on albumin-heparin and albumin microspheres suggested negative cooperativity, while positive cooperativity was observed for chicken egg lysozyme adsorption on albumin-heparin microspheres.\ud \ud Human lysozyme loading of albumin-heparin microspheres was 3 times higher than with albumin microspheres, with long term release occurring via an ion exchange mechanism. Apparent diffusion coefficients of 2.1 × 10-1 and 3.9 × 10-11cm2/sec were obtained for the release of human lysozyme from albumin-heparin and albumin microspheres, respectively. The release was found to be independent of diffusion, since the rate determining step was likely an adsorption/desorption processes. An apparent diffusion coefficient of 4.1 × 10-12 cm2/sec was determined for the release of chicken egg lysozyme from albumin-heparin microspheres.\ud \ud Low release of the lysozymes from albumin-heparin microspheres was observed in deionized water, consistent with the proposed ion exchange release mechanism. Overall, albumin-heparin microspheres demonstrated enhanced ion exchange characteristics over albumin microspheres

Release of macromolecules from albumin-heparin microspheres

Hydrophilic microspheres based on albumin-heparin conjugates have been prepared as a macromolecular delivery system. The soluble albumin-heparin conjugate was synthesized and crosslinked in a water-in-oil emulsion with glutaraldehyde to form microspheres in the same manner as for albumin microsphere preparation. The microspheres were characterized in terms of their size and swelling properties. The loading of macromolecules into albumin-heparin microspheres was carried out concurrently and after microsphere preparation. FITC-dextran was applied as a model macromolecule. A higher loading content was achieved when loading was carried out concurrently with microsphere preparation than when loaded subsequently. Prolonged release of FITC-dextran from albumin-heparin microspheres was achieved and attributed to the high molecular weight of the macromolecule. The release of FITC-dextran was modulated by crosslinking density, loading content and the method of drug incorporation. Apparently, the mechanism of FITC-dextran release from albumin-heparin microspheres was dependent on the method of drug incorporation. For release of FITC-dextran from the microspheres, assuming negligible interactions, a diffusion coefficient of 1.7 × 10¿9 cm2/s was determined