Alternating bioactivity of polymeric layer-by-layer assemblies: anticoagulation vs procoagulation of human blood

The layer-by-layer assembly between cationic chitosan and anionic dextran sulfate was analyzed quantitatively by a quartz crystal microbalance technique in the absence and presence of 0.2, 0.5, and 1 M NaCl in the polymer solution. The apparent film thickness increased upon increasing the NaCl concentration. The anti- versus procoagulant activity of these films against whole human blood was studied by the immersion of a substrate into blood for 30 min incubation time at 37 degrees C. The substrate was coated with films of varying NaCl concentrations and assembly step numbers. There was a critical concentration for the alternating activity; above a concentration of 0.5 M NaCl, both anti- and procoagulation could be observed on the dextran sulfate and chitosan surfaces, respectively. The underlying layer of the assembly was necessary for this alternating activity; after a five-step assembly, the activity was realized. The adsorption of a cationic dye (methylene blue) onto the films revealed that the anionic-charge density derived from dextran sulfate on the film surface was linearly increased with increased NaCl concentration. There was a critical charge density of the dextran sulfate for the anticoagulant activity. An assembly was also constructed from a combination of chitosan and heparin, but the activity was different from that of the former system; strong anticoagulant activity was observed even on the chitosan surface. We suggest that the polymer species and/or the assembly conditions are key factors for realizing the alternating bioactivities of films prepared by the layer-by-layer assembly.     

A study of dextran production from maltodextrin by cell suspensions of Gluconobacter oxydans NCIB 4943

This study investigated dextran synthesis from a commercial maltodextrin substrate using cell suspensions of G. oxydans NCIB 4943 as catalysts. Experiments were arranged according to a central composite statistical design. The effects of substrate concentration (10-100 g l-1), cell concentration (0.32-32.0 g wet weight l-1), time of reaction (8-48 h) and pH (3.5-5.5), each at three levels, on dextran yield and dextran molecular weight (MW), were investigated. Response surface methodology was used to assess factor interactions, and empirical models describing the two responses were fitted. Most of the variance in dextran yield could be explained by the fitted model (R2 = 0.96). Dextran yield ranged from 1.21 to 41.69%. The presence of significant negative quadratic effects of cell concentration and time indicated that dextran yield reached a plateau and thus, optimum levels of cell concentration and time could be identified to maximize dextran yield. Dextran MW ranged from 6.6 to 38 kDa and was characterized by the significant interactions of reaction time with substrate concentration and cell concentration. The model, however, could account for only 60% of the variance in dextran MW. Possible reasons for this are discussed.     

A Simple Method to Functionalize PCL Surface by Grafting Bioactive Polymers Using UV Irradiation

Background

Polycaprolactone (PCL) is a biodegradable polymer which is used in tissue engineering applications thanks to its many favorable characteristics. However, PCL surfaces are known as hydrophobic leading to a lack of favorable cell response. To overcome this problem, PCL surfaces will undergo a surface functionalization by grafting bioactive polymers bearing ionic groups.

PEM Anchorage on Titanium Using Catechol Grafting

Background

This study deals with the anchorage of polyelectrolyte films onto titanium surfaces via a cathecol-based linker for biomedical applications.

Methodology

The following study uses a molecule functionalized with a catechol and a carboxylic acid: 3-(3,4-dihydroxyphenyl)propanoic acid. This molecule is anchored to the TiO₂ substrate via the catechol while the carboxylic acid reacts with polymers bearing amine groups. By providing a film anchorage of chemisorption type, it makes possible to deposit polyelectrolytes on the surface of titanium.

Principal Findings

Infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), contact angle and atomic force microscopy (AFM) measurements show that the different steps of grafting have been successfully performed.

Conclusions

This method based on catechol anchorage of polyelectrolytes open a window towards large possibilities of clinical applications.     

Radical graft polymerization of styrene sulfonate on poly(ethylene terephthalate) films for ACL applications: "grafting from" and chemical characterization

The purpose of this study is to develop a reliable method of functionalizing poly(ethylene terephthalate) with bioactive polymers to produce a "biointegrable" artificial anterior cruciate ligament. Radical graft polymerization of the sodium salt of styrene sulfonate (NaSS) onto poly(ethylene terephthalate) (PET) films was performed using the "grafting from" technique. Prior to the grafting, the surfaces of poly(ethylene terephthalate) films were activated by ozonation to generate peroxide and hydroperoxide reactive species on the PET film surfaces. The radical polymerization of NaSS was initiated by thermal decomposition of the hydroperoxides. The grafted PET surfaces were characterized by a toluidin blue colorimetric method, X-ray photoelectron spectroscopy, contact angle measurements, and atomic force microscopy. The influence of ozonation time, monomer concentration, and temperature on NaSS grafting ratios was examined. A total of 30 min of ozonation followed by grafting from a 15% NaSS solution at 70 degrees C for 90 min or more resulted in attachment of poly(NaSS) chains to the PET film surfaces.     

Dextran Nanoparticle Synthesis and Properties

Dextran is widely exploited in medical products and as a component of drug-delivering nanoparticles (NPs). Here, we tested whether dextran can serve as the main substrate of NPs and form a stable backbone. We tested dextrans with several molecular masses under several synthesis conditions to optimize NP stability. The analysis of the obtained nanoparticles showed that dextran NPs that were synthesized from 70 kDa dextran with a 5% degree of oxidation of the polysaccharide chain and 50% substitution with dodecylamine formed a NP backbone composed of modified dextran subunits, the mean diameter of which in an aqueous environment was around 100 nm. Dextran NPs could be stored in a dry state and reassembled in water. Moreover, we found that different chemical moieties (e.g., drugs such as doxorubicin) can be attached to the dextran NPs via a pH-dependent bond that allows release of the drug with lowering pH. We conclude that dextran NPs are a promising nano drug carrier.     

Competitive protein adsorption on polysaccharide and hyaluronate modified surfaces

Adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) was measured on six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and Streptococcus zooepidemicus. Film thickness and surface morphology depended on the HA molecular weight and concentration. BSA coverage was enhanced on surfaces in competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of HA utilized. With changing bulk protein concentration from 20 to 40 μg ml^?1 for each species, Fg coverage on silicon increased by 4x, whereas both BSA and Fg adsorption on dextran and HA were far less dependent on protein bulk concentration.     

Membrane-membrane interactions: parallel membranes or patterned discrete contacts.

Theoretical and experimental studies of thin liquid films show that, under certain conditions, the film thickness can undergo a sudden transition which gives a stable narrower film or ends in film rupture at spatially periodic points. Theoretical analysis have also indicated that similar transitions might arise in the thin aqueous layer separating interacting membranes. Experiments described here show spatially periodic intermembrane contact points and suggest that spontaneous rapid growth of fluctuations can occur on an intermembrane water layer. Normal and pronase pretreated erythrocytes were exposed to 2% Dextran (450,000 Mr) and the resultant aggregates were examined by light and transmission electron microscopy. Cell electrophoresis measurements were used as an index of pronase modification of the glycocalyx. Erythrocytes exposed to dextran revealed a uniform intercellular separation of parallel membranes. This equilibrium between attractive and repulsive intermembrane forces is consistent with the established Derjaguin, Landau, Verwey, Overbeek (DLVO) model for colloidal particle interaction. In contrast to the above uniform separation a spatial pattern of discrete contact regions was observed in cells coming together in dextran following pronase pretreatment. The lateral contact separation distance was 3.0 microns for mild pronase pretreatment and decreased to 0.85 micron for more extensive pronase pretreatments. The system examined here is seen as a useful experimental model in which to study the principles involved in producing either uniform separation or point contacts between interacting membranes.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5–2.0% w/v) concentrations of Dextran T500. At concentrations of 5–7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5–2.0% w/v) concentrations of Dextran T500. At concentrations of 5–7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

Competitive protein adsorption on polysaccharide and hyaluronate modified surfaces

Adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) was measured on six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and Streptococcus zooepidemicus. Film thickness and surface morphology depended on the HA molecular weight and concentration. BSA coverage was enhanced on surfaces in competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of HA utilized. With changing bulk protein concentration from 20 to 40 μg ml(-1) for each species, Fg coverage on silicon increased by 4x, whereas both BSA and Fg adsorption on dextran and HA were far less dependent on protein bulk concentration.     

Desulfurization with <Emphasis Type="Italic">Thialkalivibrio versutus</Emphasis> immobilized on magnetic nanoparticles modified with 3-aminopropyltriethoxysilane

Objective

Thialkalivibrio versutus D301 cells were immobilized on Fe₃O₄ nanoparticles (NPs) synthesized by an improved chemical coprecipitation method and modified with 3-aminopropyltriethoxysilane (APTES), then the immobilized cells were used in sulfur oxidation.

Results

The prepared Fe₃O₄–APTES NPs had a narrow size distribution (10 ± 2 nm) and were superparamagnetic, with a saturation magnetization of 60.69 emu/g. Immobilized cells had a saturation magnetization of 34.95 emu/g and retained superparamagnetism. The optimum conditions for cell immobilization were obtained at pH 9.5 and 1 M Na⁺. The immobilization capacity of Fe₃O₄–APTES NPs was 7.15 g DCW/g-NPs that was 2.3-fold higher than that of Fe₃O₄ NPs. The desulfurization efficiency of the immobilized cells was close to 100%, having the same sulfur oxidation capacity as free cells. Further, the immobilized cells could be reused at least eight times, retaining more than 85% of their desulfurization efficiency.

Conclusion

Immobilization of cells with the modified magnetic NPs efficiently increased cell controllability, have no effect on their desulfurization activity and could be effectively used in large-scale industrial applications.

     

Interaction between dextran and human low density lipoproteins (LDL) observed using laser light scattering

Dextran infusions in humans lead to a reduction of low density lipoproteins (LDL) in the plasma compartment. The interaction of dextran with human LDL was investigated in vitro by static and dynamic light scattering. The experiments were performed with human LDL (apoB concentration 0.75 g l⁻¹) and dextran (M_w=40 000 and 70 000 g mol⁻¹) at 25°C. The dextran concentrations after mixing were 10 and 50 g l⁻¹. The hydrodynamic radius for native LDL was found to be R_H=12.9 nm. The addition of dextran induces the formation of LDL associates with a mean radius of R_H≈200 nm. These findings show that even non-sulphated polysaccharides interact with LDL. The dextran-dependent formation of LDL associates detected in vitro could be the reason for the in vivo effect of dextran on the lipid metabolism.     

Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents

Background

Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur.

Methods

Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone.

Results

Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde–dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes.

Conclusions and general significance

Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents.     

Improved antifouling properties of photobioreactors by surface grafted sulfobetaine polymers

To improve the antifouling (AF) properties of photobioreactors (PBR) for microalgal cultivation, using trihydroxymethyl aminomethane (tris) as the linking agent, a series of polyethylene (PE) films grafted with sulfobetaine (PE-SBMA) with grafting density ranging from 23.11 to 112 μg cm^?2 were prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). It was found that the contact angle of PE-SBMA films decreased with the increase in the grafting density. When the grafting density was 101.33 μg cm^?2, it reached 67.27°. Compared with the PE film, the adsorption of protein on the PE-SBMA film decreased by 79.84% and the total weight of solid and absorbed microalgae decreased by 54.58 and 81.69%, respectively. Moreover, the transmittance of PE-SBMA film recovered to 86.03% of the initial value after cleaning, while that of the PE film recovered to only 47.27%. The results demonstrate that the AF properties of PE films were greatly improved on polySBMA-grafted surfaces.     

Evidence of protein collective motions on the picosecond timescale

We investigate the presence of structural collective motions on a picosecond timescale for the heme protein, cytochrome c, as a function of oxidation and hydration, using terahertz (THz) time domain spectroscopy and molecular dynamics simulations. The THz response dramatically increases with oxidation, with the largest increase for lowest hydrations, and highest frequencies. For both oxidation states the THz response rapidly increases with hydration saturating above ∼25% (g H₂O/g protein). Quasiharmonic vibrational modes and dipole-dipole correlation functions were calculated from molecular dynamics trajectories. The collective mode density of states alone reproduces the measured hydration dependence, providing strong evidence of the existence of these motions. The large oxidation dependence is reproduced only by the dipole-dipole correlation function, indicating the contrast arises from diffusive motions consistent with structural changes occurring in the vicinity of buried internal water molecules. This source for the observed oxidation dependence is consistent with the lack of an oxidation dependence in nuclear resonant vibrational spectroscopy measurements.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5-2.0% w/v) concentrations of Dextran T500. At concentrations of 5-7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

Restoration of radiation-induced G1-depression in transformed fibroblasts treated with dextran sulfate

3T6 and 3T3 cells were cultured with dextran sulfate and irradiated with a dose of 1 000 R of ⁶⁰Co gamma-rays. The rate of progress of cells from G1 to S phase was estimated by radioautograms using ³H-thymidine as a tracer. When cultured in normal medium, 3T3 cells showed a rate of progress from G1 to S phase which was retarded by gamma-ray radiation, whereas 3T6 cells were unaffected. Dextran sulfate alone did not prolong the cell cycle time during logarithmic growth in either cell line, but reduced markedly the saturation density of 3T6 cells. Radiation-induced G1-suppression was observed in 3T6 cells which were cultured in the presence of dextran sulfate for at least 2 days. Replacement of normal media by media containing dextran sulfate at the confluent stage led to the onset of DNA synthesis (and subsequently cell division) in 3T6 cells. Gamma-ray irradiation before the change of media delayed the onset of DNA synthesis.     

Semi-quantitative assessments of dextran toxicity on corneal endothelium: conceptual design of a predictive algorithm

Dextran is added to corneal culture medium for at least 8 h prior to transplantation to ensure that the cornea is osmotically dehydrated. It is presumed that dextran has a certain toxic effect on corneal endothelium but the degree and the kinetics of this effect have not been quantified so far. We consider that such data regarding the toxicity of dextran on the corneal endothelium could have an impact on scheduling and logistics of corneal preparation in eye banking. In retrospective statistic analyses, we compared the progress of corneal endothelium (endothelium cell loss per day) of 1334 organ-cultured corneal explants in media with and without dextran. Also, the influence of donor-age, sex and cause of death on the observed dextran-mediated effect on endothelial cell counts was studied. Corneas cultured in dextran-free medium showed a mean endothelium cell count decrease of 0.7% per day. Dextran supplementation led to a mean endothelium cell loss of 2.01% per day; this reflects an increase by the factor of 2.9. The toxic impact of dextran was found to be time dependent; while the prevailing part of the effect was observed within the first 24 h after dextran-addition. Donor age, sex and cause of death did not seem to have an influence on the dextran-mediated toxicity. Based on these findings, we could design an algorithm which approximately describes the kinetics of dextran-toxicity. We reproduced the previously reported toxic effect of dextran on the corneal endothelium in vitro. Additionally, this is the first work that provides an algorithmic instrument for the semi-quantitative calculation of the putative endothelium cell count decrease in dextran containing medium for a given incubation time and could thus influence the time management and planning of corneal transplantations.     

Quantitation of dextran 70 in peritoneal dialysate from patients administered 7.5% polyglucose

A method using gel permeation chromatography was evaluated for the quantitation of dextran 70 in dialysate samples containing polyglucose. Dialysate samples containing dextran 70 and polyglucose were pretreated using the enzyme α-amylase to selectively hydrolyze the α(1–4)-linked polyglucose, while leaving the α(1–6)-linked dextran 70 intact. Following sample deproteinization with trichloroacetic acid, dextran 70 was quantitated using gel permeation chromatography with refractive index detection. This method was evaluated for accuracy, precision, specificity, linearity, range, and analyte stability. Adequate method linearity with a correlation of >0.999 was established over the range of dextran 70 concentration from 1 to 0.025 mg/ml. Method precision was approximately 2% R.S.D. and accuracy (% recovery) was approximately 98–100% in the typical sample concentration range (1–0.5 mg/ml). This method was applied to the determination of intraperitoneal fluid kinetics in continuous ambulatory peritoneal dialysis (CAPD) patients administered daily night-time intraperitoneal exchanges with either 7.5% polyglucose or 4.25% dextrose. Dextran 70 was added to the dialysis solutions to yield an initial concentration of 1 mg/ml. Dialysate samples were collected at various times over a 10-h dwell-time and assayed for dextran 70. Intraperitoneal volume profiles based on dextran 70 concentrations and drain volumes were then calculated for each dialysis solution.