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PubMed1996

In vitro measurements of inertial cavitation thresholds in human blood.

Inertial cavitation thresholds were measured in human blood exposed to pulsed ultrasound. Freshly drawn blood, bank blood and aqueous dilutions of both were used in this experimental study. Micrometer-sized polystyrene particles were used as extra potential nuclei in some samples. Focused transducers with megahertz center frequencies (2.5 MHz, 4.3 MHz) were employed to generate pulsed ultrasound to induce cavitation. Specially designed cells for hosting the blood samples were made to adapt to the experimental environment. Cavitation threshold measurements were achieved by using an active cavitation detection scheme which utilizes a highly focused transducer with a much higher center frequency (30 MHz). In 50% diluted blood samples, when no polystyrene particles were added to the samples, the threshold for cavitation was about 4.1 MPa at 2.5 MHz, while no cavitation was detected at 4.3 MHz. Generally, the measured thresholds decrease in samples with lower volume concentration of red blood cells or when polystyrene particles were added to the samples. Results show that the measured thresholds in some circumstances are in the range of output pressure of diagnostic ultrasound instrumentation; but for whole, freshly drawn blood, our apparatus was unable to detect cavitation, even at 6.3 MPa.

Blood Cells↗

PubMed2005

Conformational analysis of heparin binding peptides.

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339-364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.

Amino Acid Sequence↗

PubMed2004

Surface attachment of nanoparticles using oligonucleotides.

Colloidal polymer particles are widely used in a variety of applications ranging from chromatography to surface modified bioreactors in protein arrays. In the present study, surface attachment of polystyrene particles to a polystyrene substrate has been performed using oligonucleotide hybridization. Thiolated complementary oligomers of cytosine and guanine have been covalently coupled to a pyridyl disulphide (PDS) modified polyethyleneglycol tether, forming part of a triblock copolymer which is adsorbed to the polystyrene surfaces via hydrophobic polypropylene oxide center blocks. The ability to withstand shear forces was studied using a laminar flow cell and the uptake of oligomers on the particles was quantified using two complementary techniques: UV-spectroscopy and sedimentation field flow fractionation. The possibility to tether particles in a flow cell suitable for practical use in e.g. a FIA-system is demonstrated.

Binding Sites↗

PubMed2006

Micropatterning of proteins on nanospheres.

Currently micropatterning of proteins is mainly carried out on a planar substrate, which involves multi-step surface modifications directly on the substrate. Efficiency of chemical reactions is usually low, resulting in low signal-to-noise (S/N) ratio and poor repeatability of results. Here we presented a micropatterning method using polystyrene nanospheres with non-planar surface as a solid support for attaching proteins, which introduces many advantages. The patterning of proteins was carried out in two approaches: one was to dispense polystyrene nanospheres into an array of microwells and then attach proteins onto the nanospheres, and another was to coat polystyrene nanospheres with proteins first and then deposit the spheres into the microwells. For both approaches, a uniform pattern of proteins was generated. The amount of proteins attached via nanospheres was much higher than that on planar surface.

Fluorescein-5-isothiocyanate↗

PubMed2005

Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model.

Current vaccine development includes optimization of antigen delivery to antigen presenting cells, such as dendritic cells (DC). Particulate systems have attracted increasing attention in the development of vaccine delivery systems. In the present study, we investigated DC uptake of model fluorescent polystyrene particles with a broad size range and variable surface properties. Localization of particles was investigated using confocal laser scanning microscopy and uptake was quantified by flow cytometry. Immature DC were generated from mononuclear cells isolated from human blood. The polystyrene particles interacted with the DC throughout the tested diameter range of 0.04-15 microm in a time- and concentration-dependent manner. The optimal particle diameter for fast and efficient acquisition by a substantial percentage of the DC was 0.5 microm and below. The surface of 1 and 0.1 microm polystyrene particles was covalently modified with different polyaminoacids/proteins, yielding particles with varying surface charge. Uptake of 1 microm particles was greatly enhanced when particles displayed a positive surface charge. In general, the present findings establish that particle diameters of 0.5 microm and below were optimal for DC uptake; however uptake of larger particles could be greatly enhanced by rendering the particle surface positive. Whether increased particle uptake is correlated with increased immune responses, remains to be established.

Chemical Phenomena↗

PubMed2005

Collagen adsorption and structure on polymer surfaces observed by atomic force microscopy.

The structure and adsorption patterns of type I and type III collagen were imaged on various polymer substrates with atomic force microscopy. Type I collagen had higher adsorption on polystyrene than on a series of polymethacrylates and formed a network of tightly, interwoven strands. Upon adsorption to different polymethacrylates, with varying side chain lengths, the collagen molecules formed long, branching fibrils. Types I and III collagen had different adsorption patterns, in some cases, on the identical substrate material. For example, instead of forming a tightly packed network, type III forms long, branching fibers on the polystyrene surface. On other materials, such as poly(n-butyl methacrylate), the two types of collagen showed similar adsorption pattern and structure. Adsorbed collagen was also imaged on various blends of polystyrene and polymethacrylates to determine how the polymer surface chemical structure and surface topography mediates protein adsorption.

Adsorption↗

PubMed2005

Termination and side-reactions of polystyryllithium with beta-bromostyrene.

Low molecular weight polystyryllithium was synthesised and terminated with a two-fold molar excess of beta-bromostyrene to produce an end-functionalised polymer. 1,3-Diphenylallyl-terminated polystyrene and other reaction products were analysed by FT-Raman spectroscopy. By comparison with model compounds, it can be deduced that beta-bromostyrene with a predominantly trans configuration gave rise to primarily cis conformational unsaturation at the polystyrene chain-end. It was also demonstrated that a polystyrene 'dimer' and 1,4-diphenylbutadiene were produced via side-reactions which are considered to result from metal-halogen exchange in the reagents.

Kinetics↗

PubMed2006

Poly-L-lysine-coated nanoparticles: a potent delivery system to enhance DNA vaccine efficacy.

DNA formulations provide the basis for safe and cost efficient vaccines. However, naked plasmid DNA is only poorly immunogenic and new effective delivery strategies are needed to enhance the potency of DNA vaccines. In this study, we present a novel approach for the delivery of DNA vaccines using inert poly-L-lysine (PLL) coated polystyrene particles, which greatly enhance DNA immunogenicity. Intradermal injection of plasmid DNA encoding for chicken egg ovalbumin (OVA) complexed with PLL-coated polystyrene nanoparticles induced high levels of CD8 T cells as well as OVA-specific antibodies in C57BL/6 mice and furthermore inhibited tumour growth after challenge with the OVA expressing EG7 tumour cell line. Importantly, vaccine efficacy depended critically on the size of the particles used as well as on the presence of the PLL linker. Our data show that PLL-coated polystyrene nanoparticles of 0.05 microm but not 0.02 microm or 1.0 microm in diameter are highly effective for the delivery of DNA vaccines.

Adjuvants, Immunologic↗

PubMed1999

Protein adhesion force dynamics and single adhesion events.

Using the manipulation force microscope, a novel atomic force microscope, the adhesion forces of bovine serum albumin, myoglobin, ferritin, and lysozyme proteins to glass and polystyrene substrates were characterized by following the force necessary to displace an adsorbed protein-covered microsphere over several orders of magnitude in time. This force was consistent with a power law with exponent a = 0.37 +/- 0.03 on polystyrene, indicating that there is no typical time scale for adhesion on this substrate. On glass, the rate of adhesion depended strongly on protein charge. Forces corresponding to single protein adhesion events were identified. The typical rupture force of a single lysozyme, ferritin, bovine serum albumin, and myoglobin protein adhering to glass was estimated to be 90 +/- 10 pN, 115 +/- 13 pN, 277 +/- 44 pN, and 277 +/- 44 pN, respectively, using a model of the experimental system. These forces, as well as the force amplitudes on hydrophobic polystyrene, correlate with protein stiffness.

Animals↗

PubMed2002

Temperature gradient interaction chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of air terminated polystyryllithium.

The reaction products of polystyryllithium with air were characterized by size-exclusion chromatography, temperature gradient interaction chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Polystyryllithium was prepared by anionic polymerization of styrene initiated with sec-butyllithium in cyclohexane under an Ar atmosphere. It was confirmed that polystyryl ketone, polystyryl alcohol, and directly coupled polystyrene were the major products in addition to the normally terminated polystyrene, which is consistent with the results in the literature. We could also identify the presence of methoxy and carboxylic acid end capped polystyrenes as well as dipolystyryl ether as minor products. Among the minor products, dipolystyryl ether has not been reported yet.

Air↗

PubMed1995

Growth and fibrinolytic parameters of human umbilical vein endothelial cells seeded onto cardiovascular grafts.

It was the aim of this study to investigate possible effects of biomaterials used to produce vascular grafts on the fibrinolytic system of endothelial cells. Therefore growth conditions for human umbilical vein endothelial cells on polytetrafluoroethylene and on polyurethane were optimized. Tissue culture polystyrene was used as a control material. We could demonstrate that precoating of the materials with fibronectin significantly increased the growth rate of human umbilical vein endothelial cells on these materials. Furthermore, we showed that human umbilical vein endothelial cells grown on polytetrafluoroethylene or polyurethane released more plasminogen activator inhibitor-1 and tissue type-plasminogen activator into the conditioned media than did human umbilical vein endothelial cells grown on tissue culture polystyrene. Human umbilical vein endothelial cells cultured on polytetrafluoroethylene also deposited more plasminogen activator inhibitor-1 into the extracellular matrix than did control cells grown on tissue culture polystyrene. Our results give evidence that human umbilical vein endothelial cells grown on two biomaterials used to construct vascular grafts, namely polytetrafluoroethylene and polyurethane, produce tissue-type plasminogen activator as well as plasminogen activator inhibitor-1, two major components of the fibrinolytic system also expressed by endothelial cells in vivo. In conclusion, our data suggest that endothelial cells grown on vascular grafts show functional integrity concerning their fibrinolytic system, which in turn might contribute to reduce the thrombogenic properties of the graft material.

Biocompatible Materials↗

PubMed1998

Behaviour of human osteoblasts cultured on bioactive glass coatings.

Two new formulations of bioactive glasses were used as coatings on titanium alloy (TiAl6V4) implants for prosthetic applications in the orthopaedic field. The biocompatibility of these bioglasses, as well as their osteoconductive properties, were assessed by employing primary cultures of human osteoblasts. A nonbioactive glass, the titanium alloy and polystyrene surface were used as controls. The results obtained demonstrated that the two bioglasses elicited a rapid and strong proliferative response by osteoblasts, which spread, formed a close layer and then expressed the specific osteoblastic marker i.e. osteocalcin. In comparison, cells grew on the nonbioactive glass to a much minor extent, similar to that of polystyrene control, showing individual cellular elements not forming a compact sheet, but expressed levels of osteocalcin clearly higher than both the polystyrene control and the two bioglasses. Finally, a very low proliferative rate of osteoblasts and the synthesis of hardly detectable osteocalcin amounts were observed with the titanium alloy. In conclusion, our studies indicate that the new bioactive glasses are effective in stimulating osteoblast growth and differentiation.

Alloys↗

PubMed1999

Enhanced proliferation and osteocalcin production by human osteoblast-like MG63 cells on silicon nitride ceramic discs.

The biocompatibility of silicon nitride (Si3N4) was assessed in an in vitro model using the human osteoblast-like MG-63 cell line. Cells were propagated on the surface of: reaction-bonded silicon nitride discs, sintered after reaction-bonded silicon nitride discs or control polystyrene surface for 48 h. Compared to cells propagated on polystyrene surface, cells grown on the surface of unpolished silicon nitride discs had significantly lower cell yield and decreased osteocalcin production. In contrast, cells on the surface of polished silicon nitride discs showed similar proliferative capacity to control cells propagated on polystyrene surface. Cells propagated on polished discs also produced higher levels of osteocalcin than cells on unpolished discs. SEM analysis showed cells with well-delineated morphology and cytoplasmic extensions when propagated on polished sintered after reaction-bonded discs. Cells appeared more spherical, when grown on polished reaction-bonded discs. The results of this study suggest that silicon nitride is a non-toxic, biocompatible ceramic surface for the propagation of functional human bone cells in vitro. Its high wear resistance and ability to support bone cell growth and metabolism make silicone nitride an attractive candidate for clinical application. Further studies are needed to explore the feasibility of using silicon nitride clinically as an orthopedic biomaterial.

Cell Culture Techniques↗

PubMed2002

Chemical camouflage of nanospheres with a poorly reactive surface: towards development of stealth and target-specific nanocarriers.

A two-step approach is described to chemically camouflage the inert surface of model polystyrene nanospheres of 60 nm in diameter against recognition by the body's defenses. The first step was based on the strong protein adsorbing potency of polystyrene, and the second step utilized the chemical reactivity of the adsorbed proteins for conjugation with cyanuric chloride-activated methoxypoly(ethyleneglycol)5000, mPEG5000. Bovine serum albumin (BSA) and rat IgG were used as models of non-immune and immune proteins, respectively. The maximum adsorbance values for both proteins were near expectation for a close-packed monolayer. Adsorption isotherms studies and analysis of the hydrodynamic thickness of the adsorbed protein layer confirmed the close-packed side-on mode of adsorption for BSA and the end-on mode of adsorption for IgG, respectively. Nucleophiles on the adsorbed proteins were then reacted with cyanuric chloride activated mPEG5000. The average poly(ethyleneglycol) (PEG) content for a 60-nm nanospheres was found to be 13.7+/-0.4 micromol PEG/micromol BSA and 3.6+/-0.3 micromol PEG/micromol IgG. The interaction of both PEG-bearing nanospheres with the hydrophobic column material octyl-agarose indicated surface heterogeneity among the nanospheres. Only nanospheres with the most hydrophilic phenotype (approximately 70% of the total population) exhibited stealth properties after intravenous injection to rats. In contrast to the described approach, incubation of uncoated nanospheres with preformed BSA-mPEG5000 conjugates failed to produce long circulating entities. For design of splenotropic particles cyanuric chloride-activated mPEG5000 was conjugated to BSA-coated polystyrene beads of 225 nm in diameter. Despite their stealth property to hepatic Kupffer cell recognition, these nanospheres were cleared by the splenic red pulp macrophages.

Adsorption↗

PubMed2003

Sample metallization for performance improvement in desorption/ionization of kilodalton molecules: quantitative evaluation, imaging secondary ion MS, and laser ablation.

The metallization procedure, proposed recently for signal improvement in organic secondary ion mass spectrometry (SIMS) (Delcorte, A.; Médard, N.; Bertrand, P. Anal.Chem. 2002, 74, 4955)., has been thoroughly tested for a set of kilodalton molecules bearing various functional groups: Irganox 1010, polystyrene, polyalanine, and copper phthalocyanine. In addition to gold, we evaluate the effect of silver evaporation as a sample treatment prior to static SIMS analysis. Ion yields, damage cross sections, and emission efficiencies are compared for Ag- and Au-metallized molecular films, pristine coatings on silicon, and submonolayers of the same molecules adsorbed on silver and gold. The results are sample-dependent but as an example, the yield enhancement calculated for metallized Irganox films with respect to untreated coatings is larger than 2 orders of magnitude for the quasimolecular ion and a factor of 1-10 for characteristic fragments. Insights into the emission processes of quasimolecular ions from metallized surfaces are deduced from kinetic energy distribution measurements. The advantage of the method for imaging SIMS applications is illustrated by the study of a nonuniform coating of polystyrene oligomers on a 100-microm polypropylene film. The evaporated metal eliminates sample charging and allows us to obtain enhanced quality images of characteristic fragment ions as well as reasonably contrasted chemical mappings for cationized PS oligomers and large PP chain segments. Finally, we report on the benefit of using metal evaporation as a sample preparation procedure for laser ablation mass spectrometry. Our results show that the fingerprint spectra of Au-covered polystyrene, polypropylene, and Irganox films can be readily obtained under 337-nm irradiation, a wavelength for which the absorption of polyolefins is low. This is probably because the gold clusters embedded in the sample surface absorb and transfer the photon energy to the surrounding organic medium.

Butylated Hydroxytoluene↗

PubMed2007

Hydration and energy dissipation measurements of biomolecules on a piezoelectric quartz oscillator by admittance analyses.

By using a 27-MHz piezoelectric quartz oscillator connected with a vector network analyzer, we obtained resonance frequency decreases (-DeltaFwater) and energy dissipation increases (DeltaDwater) during binding of biotinylated bovine serum albumin, biotinylated ssDNA, biotinylated dsDNA, and biotinylated pullulan to a NeutrAvidin-immobilized 27-MHz quartz crystal microbalance (QCM) plate in aqueous solution, as well as in the wet air phase (98% humidity, -DeltaFwet and DeltaDwet) and in the dry air phase (-DeltaFair and DeltaDair). -DeltaFwater indicates the total mass of the molecule, bound water, and vibrated water in aqueous solutions. -DeltaFwet indicates the total mass of the molecule and bound water. -DeltaFair simply shows the real mass of the molecule on the QCM. In terms of results, (-DeltaFwet)/(-DeltaFair) values indicated the bound water ratios per unit biomolecular mass were on the order of pullulan (2.1-2.2) > DNAs = proteins (1.4-1.6) > polystyrene (1.0). The (-DeltaFwater)/(-DeltaFair) values indicated the hydrodynamic water (bound and vibrated water) ratios per unit biomolecular mass were on the order of dsDNA (6.5) > ssDNA = pullulan (3.5-4.4) > proteins (2.4-2.5) > polystyrene (1.0). Energy dissipation parameters per unit mass in water (DeltaDwater/(-DeltaFair)) were on the order of pullulan > dsDNA > ssDNA > proteins > polystyrene. Energy dissipation in the wet and dry air phases (DeltaDwet and DeltaDair) were negligibly small, which indicates even these biomolecules act as elastic membranes in the air phase (without aqueous solution). We obtained a good linear relationship between [(-DeltaFwater)/(-DeltaFair) - 1], which is indicative of hydration and DeltaDwater/(-DeltaFair) of proteins. The aforementioned values suggest that the energy dissipation of proteins was mainly caused by hydration and that proteins themselves are elastic molecules without energy dissipation in aqueous solutions. On the contrary, plots in cases of denatured proteins, DNAs, and pullulans were relatively deviant toward the large hydration and energy dissipation from the theoretical line as perfect elastic materials, meaning that the large energy dissipation occurs because of viscoelastic properties of denatured proteins, linear DNAs, and pullulans in the water phase, in addition to energy dissipation due to the hydration of molecules. These two parameters could characterize various biomolecules with structural properties in aqueous solutions.

Air↗

PubMedDate not supplied

Sustained high-yield production of recombinant proteins in transiently transfected COS-7 cells grown on trimethylamine-coated (hillex) microcarrier beads.

The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.

Animals↗

PubMedDate not supplied

An alkylsilyl-tethered, high-capacity solid support amenable to diversity-oriented synthesis for one-bead, one-stock solution chemical genetics.

The synthesis and use of an alkylsilyl-tethered large (500-600 microm) polystyrene resin (1) are disclosed. An optimized Suzuki coupling of bromine-functionalized polystyrene and a silicon-functionalized alkylborane generates the silicon-substituted polystyrene 1 in large scale (>100 g). Resin loading is accomplished by activation as the silyl triflate, which can accommodate even sterically encumbered secondary alcohols and phenols. Treatment with HF/pyridine for linker cleavage is mild, efficient, and amenable to an automated, large-scale distribution system. This platform delivers, minimally, 50 nmol of each small molecule derived from a diversity-oriented, split-pool synthesis on a per bead basis for use in both forward and reverse chemical genetic assays. This technology satisfies many requirements of a one bead-one stock solution approach to chemical genetics.

Alkynes↗