Method for estimating the single molecular affinity.

Affinity constants (k(d), k(a), and K(D)) can be determined by methods that apply immobilized ligands such as immunoassays and label-free biosensor technologies. This article outlines a new surface plasmon resonance (SPR) array imaging method that yields affinity constants that can be considered as the best estimate of the affinity constant for single biomolecular interactions. Calculated rate (k(d) and k(a)) and dissociation equilibrium (K(D)) constants for various ligand densities and analyte concentrations are extrapolated to the K(D) at the zero response level (K(D)(R0)). By applying this method to an LGR5-exo-Fc-RSPO1-FH interaction couple, the K(D)(R0) was determined as 3.1 nM.

Release of anti-restenosis drugs from poly(ethylene oxide)-poly(DL-lactic-co-glycolic acid) nanoparticles.

Dexamethasone- or rapamycin-loaded nanoparticles based on poly(ethylene oxide) and poly(dl-lactic-co-glycolic acid) block copolymers (PEO-PLGA) were prepared without additional stabilizer using the salting-out method. A fast release of drug in PBS (pH 7.4) at 37 degrees C resulting in 100% release within 5 h was observed for both drugs. The rate of drug release was substantially reduced by treating the particles with gelatin or albumin after drug loading, resulting in a linear drug release in time. It was shown that the rate of drug release is related to the amount of protein associated with the nanoparticles. After gelatin treatment of drug-loaded nanoparticles, sustained release of dexamethasone for 17 days and of rapamycin for 50 days could be achieved.

Biodegradable polymersomes as a basis for artificial cells: encapsulation, release and targeting.

The encapsulation of biofunctional compounds, release properties and targetability of polymersomes of amphiphilic block-copolymers based on poly(ethylene glycol) (PEG) and biodegradable polyesters or polycarbonate are described. Carboxyfluorescein (CF), as a model for hydrophilic biofunctional compounds, could be readily incorporated in the polymersomes by adding the compound to the aqueous phase during polymersome preparation. The release of encapsulated material from the polymersomes can be adjusted by changing the copolymer composition, especially the molecular weight and type of hydrophobic block of the copolymer. The presence of plasma proteins other than albumin suppressed the release of CF. CF release in PBS both at room temperature and at 60 degrees C followed first order kinetics, confirming that the CF containing polymersome system is a membrane controlled reservoir system. These biodegradable polymersomes have the potential to be targeted to specific sites in the body as shown by the specific interaction of anti-human serum albumin immobilized polymersomes with a human serum albumin coated sensor surface.

In vitro degradation of nanoparticles prepared from polymers based on DL-lactide, glycolide and poly(ethylene oxide).

Nanoparticles of poly(DL-lactic acid) (PDLLA), poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene oxide)-PLGA diblock copolymer (PEO-PLGA) were prepared by the salting-out method. The in vitro degradation of PDLLA, PLGA and PEO-PLGA nanoparticles in PBS (pH 7.4) at 37 degrees C was studied. The particle size, molecular weight of the polymers and the amount of lactic and glycolic acids formed were followed in time. PDLLA nanoparticles gradually degraded over a period of 2 years and retain their size during that period. A faster degradation was observed for PLGA nanoparticles, which was nearly complete after 10 weeks. PLGA nanoparticles retained their size during that period. In PEO-PLGA nanoparticles, the ester bond connecting the PEO and the PLGA segments was preferentially cleaved, which led to a relatively fast decrease in molecular weight and to (partial) aggregation, as multimodal size distributions were observed. PEO-PLGA nanoparticles were almost completely degraded within 8 weeks.

Polyethylene glycol-grafted polystyrene particles.

Densely pegylated particles that can serve as a model system for artificial cells were prepared by covalently grafting amino polyethylene glycol (PEG, molecular weight 3400 or 5000) onto carboxyl polystyrene particles (PS-COOH) using carbodiimide chemistry. PEG-modified particles (PS-PEG) were characterized by determination of the PEG surface concentration, zeta-potential, size, and morphology. Under optimized grafting conditions, a dense "brush-like" PEG layer was formed. A PEG surface concentration of approximately 60 pmol/cm2, corresponding with an average distance between grafted PEG chains of approximately 17 A can be realized. It was shown that grafting of PEG onto PS-COOH reduced the adsorption of proteins from human plasma (85 vol %) in phosphate-buffered saline up to 90%.

Pegylated polystyrene particles as a model system for artificial cells.

Pegylated polystyrene particles (PS-PEG) were prepared as a model system for artificial cells, by modification of carboxyl polystyrene particles (PS-COOH) with homo- and hetero-bifunctional polyethylene glycols (PEG, MW 1500, 3400, and 5000) containing an amino end group for immobilization and an amino, hydroxyl, or methoxy end group that is exposed at the surface after immobilization. Protein adsorption from human plasma dilutions (85 v %) onto PS-PEG with a PEG surface concentration higher than 40 pmol/cm2 was reduced up to 90-95% compared with protein adsorption onto PS-COOH with a final protein surface concentration of approximately 30 ng/cm2. Two-dimensional gel electrophoresis analyses showed that 30% of the total amount of adsorbed proteins onto PS-PEG are dysopsonins (i.e., nonadhesive proteins like albumin and apolipoproteins). For PS-COOH, <15% of the amount of adsorbed proteins are dysopsonins. In addition, the generation of terminal complement compound (TCC) by PS-PEG particles with a PEG surface concentration lower than approximately 55 pmol/cm2 is not significant. The low protein adsorption, the relatively high percentage of adsorbed dysopsonins, and the low level of complement activation may prevent the uptake of PS-PEG by the mononuclear phagocytic system (MPS) in vivo. Moreover, PS-PEG (PEG surface concentration > approximately 35 pmol/cm2) shows minimal interaction with cultured human umbilical vein endothelial cells (HUVEC), which mimics the endothelial lining of the blood vessel wall.

The preparation of monodisperse biodegradable polyester nanoparticles with a controlled size.

In local drug delivery, nanoparticles based on biodegradable polymers can function as vehicles with controlled drug-release properties. To achieve a well-controlled drug-release profile, control over the particle size is of great importance. Therefore, biodegradable polyester nanoparticles were prepared by the salting-out method. Process variables were varied to study the effect on the particle size. The monodisperse particles obtained were between 100 and 400 nm in size and spherical in shape. It was found that the particle size could be adjusted by varying the preparation conditions upon which the polymer concentration had the most pronounced effect.

ACPA fine-specificity profiles in early rheumatoid arthritis patients do not correlate with clinical features at baseline or with disease progression.

INTRODUCTION: Autoantibodies against citrullinated peptides/proteins (ACPA) are found in approximately 75% of the sera of patients with rheumatoid arthritis (RA). The RA-specific ACPA are frequently present prior to disease onset and their presence associates with a more erosive disease course. ACPA can therefore be used to aid the diagnosis and prognosis of RA. Recently, it became clear that ACPA are very heterogeneous, both in an individual patient and among different patients. The aim of this study was to investigate whether clinically meaningful ACPA profiles exist in early RA patients. METHODS: Twenty citrullinated peptides and the corresponding non-citrullinated control peptides were immobilized on microarray sensor chips. Sera from 374 early arthritis patients were analyzed by surface plasmon resonance imaging (iSPR) of biomolecular interactions on the sensor chip. RESULTS: Cluster analysis of the reactivities with the citrullinated peptides, after subtraction of the reactivities with the corresponding control peptides confirmed the heterogeneity of the ACPA response in RA and revealed 12 distinct ACPA profiles. The association of the 5 most frequent profiles with clinical features at diagnosis and during the disease course was examined, showing no statistically significant associations. CONCLUSIONS: Compared to the detection of ACPA in RA sera by CCP-based assays, ACPA profiling in early arthritis patients did not reveal associations with disease activity and progression scores.

Surface modification of elastomeric stamps for microcontact printing of polar inks.

Chemical modification of the surface of a stamp used for microcontact printing (microCP) is interesting for controling the surface properties, such as the hydrophilicity. To print polar inks, plasma polymerization of allylamine (PPAA) was employed to render the surface of poly(dimethylsiloxane) (PDMS), polyolefin plastomers (POP), and Kraton elatomeric stamps hydrophilic for long periods of time. A thin PPAA film of about 5 nm was deposited on the stamps, which increased the hydrophilicity, and which remained stable for at least several months. These surface-modified stamps were used to transfer polar inks by microCP. The employed microCP schemes are as follows: (a) a second generation of dendritic ink having eight dialkyl sulfide end groups to fabricate patterns on gold substrates by positive microCP, (b) fluorescent guest molecules on beta-cyclodextrin (beta-CD) printboards on glass employing host-guest recognition, and (c) Lucifer Yellow ethylenediamine resulting in covalent patterning on an aldehyde-terminated glass surface. All experiments resulted in an excellent performance of all three PPAA-coated stamp materials to transfer the polar inks from the stamp surface to gold and glass substrates by microCP, even from aqueous solutions.

Signal amplification on planar and gel-type sensor surfaces in surface plasmon resonance-based detection of prostate-specific antigen.

This article describes surface plasmon resonance (SPR)-based detection of prostate-specific antigen (PSA), comparing amplification with colloidal gold (10nm diameter) and latex microspheres (120 nm diameter) on planar- and gel-type sensor surfaces. As matrix, 3% BSA in PBS was used. Experimental data were compared with model calculations that predict the SPR signal that results from covering of the different sensor surfaces with each of the particles used. Amplification with latex particles gave a higher signal than did that with colloidal gold. However, the limit of detection (LOD) attained by latex amplification was not as good as that obtained after gold amplification, and this was unexpected. LOD and sensitivity of the amplified PSA assays when performed with the planar-type sensor disc were equally good or better compared with those when performed with the gel-type sensor disc. Indirect evidence indicates a restricted accessibility of the gel layer on the gel-type sensor toward the colloidal gold. Application of colloidal gold led to a sensitivity increase of approximately three orders of magnitude compared with nonamplified detection. The corresponding LOD was approximately 0.15 ng PSA/ml, which is sufficient for measuring enhanced, clinically relevant PSA levels (>4 ng/ml).

Enhanced bone marrow stromal cell adhesion and growth on segmented poly(ether ester)s based on poly(ethylene oxide) and poly(butylene terephthalate).

In previous studies in rats and goats, hydrophilic compositions of the PEOT/PBT block copolymer family have shown in vivo calcification and bone bonding. These copolymers are therefore interesting candidates as scaffolding materials in bone tissue engineering applications. Model studies using goat bone marrow stromal cells, however, showed that it was not possible to culture bone marrow stromal cells in vitro on these hydrophilic copolymers. In this paper two ways of surface modifying these materials to improve in vitro bone marrow stromal cell attachment and growth are discussed. Two different approaches are described: (1) blending of hydroxyapatite (HA) followed by CO(2) gas plasma etching; (2) surface modification using CO(2) gas plasma treatments. It was observed that not only HA but also the CO(2) plasma treatment by itself has a positive effect on bone marrow stromal cell attachment and growth. Gas plasma treatment appeared to be the most successful approach, resulting in a large increase in the amount of bone marrow stromal cells present on the surface (determined by a DNA assay). The amount of DNA present on the plasma-treated copolymer 1000/70/30 PEOT/PBT, based on poly(ethylene oxide, M(w) = 1000, 70 m% soft segment), was comparable to the amount present on PDLLA and significantly higher than the amount present on PCL after 7 days of cell culturing. The fact that after gas plasma treatment bone marrow stromal cells do attach to PEOT/PBT copolymers, enables in vitro bone marrow stromal cell culturing, making bone tissue engineering applications of these materials possible.

Electroosmotic guiding of sample flows in a laminar flow chamber.

The so-called address-flow principle is described: a valveless, electroosmotically driven technology used for controlling the stream profile in a laminar flow chamber. The method is explained, and a theoretical description and experimental verification are presented. Adjustment of the flow of two electroosmotically controlled guiding streams, running parallel to a central sample stream, can be used for positioning the sample stream in the dimension perpendicular to the flow direction. The results presented show that address-flow microfluidics allow easy and accurate control of sample stream position and width. The electroosmotic flow (EOF)-controlled guiding of microfluidic flows described in this paper, is a new unit operation that might aid in separation and collection in microfluidic devices. One possible application of address-flow microfluidics is guiding of capillary electrophoresis-separated components over a multisensor array, in order to perform affinity assays.