Polymeric coatings that mimic the endothelium: combining nitric oxide release with surface-bound active thrombomodulin and heparin

Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin's activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR-PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N(2)O(2))). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices.     

Poly(dimethylsiloxane)-poly(ethylene oxide)-heparin block copolymers. II: Surface characterization and in vitro assessments

Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), as well as heparin-coated glass beads and tubes were evaluated for the amounts and activities of surface-immobilized heparin. Because the amphiphilic copolymer system is thermodynamically predicted to demonstrate low-energy phase enrichment on the surfaces of air-cast films, studies were also undertaken to understand the in vitro results. Solvent-cast copolymer films have a heterogeneous microphase-separated structure according to transmission electron micrographs. Wilhelmy plate contact angle analysis indicates significant surface restructuring occurs upon hydration. Attenuated total reflectance infrared spectroscopy studies of the desiccated and hydrated films at two different sampling depths show compositional heterogeneity as a function of depth, as well as near surface restructuring allowing surface enrichment of the high-energy segments following contact with water. Significant concentrations of heparin are detected on the surface of these coatings by toluidine blue assays. In addition, a portion of the surface-bound heparin maintains its original bioactivity as determined by recalcification times, thrombin times, and Factor Xa assays. These substrates were also tested for platelet adhesion and activation reactions in vitro using polymer-coated beads in rabbit platelet-rich plasma. Heparinized polymers promoted low levels of platelet adhesion and serotonin release. Surface concentrations of heparin from bioactivity assays were then correlated with platelet adhesion and the extent of platelet release to assess the efficacy of this heparin-immobilized copolymer as a blood-compatible material or coating.     

In vitro platelet adhesion on polymeric surfaces with varying fluxes of continuous nitric oxide release

Nitric oxide (NO) is released by endothelial cells that line the inner walls of healthy blood vessels at fluxes ranging from 0.5 x 10(-10) to 4.0 x 10(-10) mol cm(-2) min(-1), and this continuous NO release contributes to the extraordinary thromboresistance of the intact endothelium. To improve the biocompatibility of blood-contacting devices, a biomimetic approach to release/generate NO at polymer/blood interfaces has been pursued recently (with NO donors or NO generating catalysts doped within polymeric coatings) and this concept has been shown to be effective in preventing platelet adhesion/activation via several in vivo animal studies. However, there are no reports to date describing any quantitative in vitro assay to evaluate the blood compatibilities of such NO release/generating polymers with controlled NO fluxes. Such a methodology is desired to provide a preliminary assessment of any new NO-releasing material, in terms of the effectiveness of given NO fluxes and NO donor amounts on platelet activity before the more complex and costly in vivo testing is carried out. In this article, we report the use of a lactate dehydrogenase assay to study in vitro platelet adhesion on such NO-releasing polymer surfaces with varying NO fluxes. Reduced platelet adhesion was found to correlate with increasing NO fluxes. The highest NO flux tested, 7.05 (+/-0.25) x 10(-10) mol cm(-2) min(-1), effectively reduced platelet adhesion to nearly 20% of its original level (from 14.0 (+/-2.1) x 10(5) cells cm(-2) to 2.96 (+/-0.18) x 10(5) cells cm(-2)) compared to the control polymer coating without NO release capability.     

A nonelutable low-molecular weight heparin stent coating for improved thromboresistance

Low-molecular weight heparin (LMWH) has been widely used as a systemic anticoagulant during percutaneous coronary intervention. In this study, LMWH was covalently immobilized to the surface of a cobalt chromium reservoir-based sirolimus-eluting stent to create a nonelutable nanoscale coating for enhanced thromboresistance. Toludine-blue stained stents revealed uniform heparin coverage on all surfaces of the stent. Scanning electron microscopy of stent strut cross-sections showed identical coating thickness on all sides; while the thickness was determined to be 320 nm by a focus-ion beam system. Secondary ion mass spectrometry showed constant concentrations of O, N, and S atoms throughout the depth of the surface, confirming the uniformity of the heparin coating. The nonelutable nature of the coating was confirmed in a modified Factor Xa inhibition assay which showed the stent had an equivalent of 3-5 heparin units/cm(2), while no elutable heparin was detected in wash solutions. The antithrombin binding capacity of the immobilized heparin was determined to be 60-80 pmol/cm(2) in an antithrombin uptake assay. The enhanced thromboresistance of the heparin coating was demonstrated in an in-vitro bovine blood flow loop which showed minimal visual thrombus accumulation and 95% reduction in platelet deposition compared to uncoated control stents. Drug-eluting stents with such nonelutable LMWH coating would represent a significant advance in the treatment of patients with complex lesions who are at increased risk of developing stent thrombosis.     

Catalytic generation of nitric oxide from nitrite at the interface of polymeric films doped with lipophilic CuII-complex: a potential route to the preparation of thromboresistant coatings

A novel approach potentially useful for the development of more thromboresistant polymeric materials is examined. The method is based on the catalytic generation of nitric oxide (NO) via Cu(I) mediated reduction of nitrite ions. Preliminary solution phase studies demonstrate that ascorbate or thiolate anions can generate Cu(I) from Cu(II) with subsequent catalytic conversion of any nitrite ions present to NO by the unstable Cu(I) species. Incorporation of this same chemistry within a hydrophobic polymeric material requires immobilizing Cu(II) ions into a polymeric phase via use of a lipophilic Cu(II) chelating ligand (dibenzo [e,k]-2,3,8,9-tetraphenyl-1,4,7,10-tetraaza-cyclododeca-1,3,7,9-tetraene (DTTCT)). It is shown that this complex can be reduced to its Cu(I) form by appropriate reducing equivalents present in the bathing solution. The resulting Cu(I) complex can then reduce nitrite to NO with the NO generation occurring at the polymer/solution interface at physiological pH. Data from chemiluminescence experiments indicate that the flux of NO at the polymer surface is comparable to that of endothelial cells (>/=1x10(-10)mol/cm(2)min) when 0.5mM nitrite/1mM ascorbate are present in the bathing solution. Potentially more useful NO generation can be achieved by doping the polymer film with the Cu(II) complex along with a lipophilic quaternary ammonium nitrite salt. In this case reducing equivalents within the aqueous phase enable the nitrite derived from the polymer to be converted into NO by the Cu(II/I) ligand complex. Films of this type are shown to generate NO for at least 6h in PBS buffer with fluxes on the order of 1.5x10(-10)mol/cm(2)min. Physiologically relevant levels of NO release are also shown to exist at the polymer interface when films are soaked in fresh plasma as well as undiluted whole blood, indicating that endogenous reducing equivalents present in blood can efficiently reduce the Cu(II)-ligand within the polymer film. The prospects of using these new NO releasing films to devise more biocompatible polymeric coatings for biomedical applications are discussed.     

Nitric oxide releasing silicone rubbers with improved blood compatibility: preparation, characterization, and in vivo evaluation

Nitric oxide (NO) releasing silicone rubbers (SR) are prepared via a three-step reaction scheme. A diamino triaminoalkyltrimethoxysilane crosslinker is used to vulcanize hydroxyl terminated polydimethylsiloxane (PDMS) in the presence of ambient moisture and a dibutyltin dilaurate catalyst so that the respective diamine triamine groups are covalently linked to the cured SR structure. These amine sites are then diazeniumdiolated, in situ, when the cured SR is reacted with NO at elevated pressure (80 psi). Although nitrite species are also formed during the NO addition reaction, in most cases the diazeniumdiolated polymer is the major product within the final SR matrix. Temperature appears to be the major driving force for the dissociation of the attached diazeniumdiolate moieties, whereas the presence of bulk water bathing the SR materials has only minimal effect on the observed NO release rate owing to the low water uptake of the SR matrices. The resulting SR films/coatings release NO at ambient or physiological temperature for up to 20 d with average fluxes of at least 4 x 10(10) mol x cm(-2) x min(-1) (coating thickness > or = 600 microm) over first 4 h, comparable to the NO fluxes observed from stimulated human endothelial cells. The NO loading and concomitant NO release flux of the SR material are readily adjustable by altering the diamine triamine loading and film/coating thickness. The new NO releasing SR materials are shown to exhibit improved thromboresistance in vivo, as demonstrated via reduced platelet activation on the surface of these polymers when used to coat the inner walls of SR tubings employed for extracorporeal circulation in a rabbit model.     

Polymers incorporating nitric oxide releasing/generating substances for improved biocompatibility of blood-contacting medical devices

The current state-of-the-art with respect to the preparation, characterization and biomedical applications of novel nitric oxide (NO) releasing or generating polymeric materials is reviewed. Such materials show exceptional promise as coatings to prepare a new generation of medical devices with superior biocompatiblity. Nitric oxide is a well-known inhibitor of platelet adhesion and activation, as well as a potent inhibitor of smooth muscle cell proliferation. Hence, polymers that release or generate NO locally at their surface exhibit greatly enhanced thromboresistivity and have the potential to reduce neointimal hyperplasia caused by device damage to blood vessel walls. In this review, the use of diazeniumdiolates and nitrosothiols as NO donors within a variety polymeric matrixes are summarized. Such species can either be doped as discrete NO donors within polymeric films, or covalently linked to polymer backbones and/or inorganic polymeric filler particles that are often employed to enhance the strength of biomedical polymers (e.g., fumed silica or titanium dioxide). In addition, very recent efforts to create catalytic polymers possessing immobilized Cu(II) sites capable of generating NO from endogenous oxidized forms of NO already present in blood and other physiological fluids (nitrite and nitrosothiols) are discussed. Preliminary literature data illustrating the efficacy of the various NO release/generating polymers as coatings for intravascular sensors, extracorporeal blood loop circuits, and arteriovenous grafts/shunts are reviewed.     

Preparation and characterization of hydrophobic polymeric films that are thromboresistant via nitric oxide release

The preparation of hydrophobic polymer films (polyurethane and poly(vinyl chloride)) containing nitric oxide (NO)-releasing diazeniumdiolate functions is reported as a basis for improving the thromboresistivity of such polymeric materials for biomedical applications. Several different approaches for preparing NO-releasing polymer films are presented, including: (1) dispersion of diazeniumdiolate molecules within the polymer matrix; (2) covalent attachment of the diazeniumdiolate to the polymer backbone; and (3) ion-pairing of a diazeniumdiolated heparin species to form an organic soluble complex that can be blended into the polymer. Each approach is characterized in terms of NO release rates and in vitro biocompatibility. Results presented indicate that the polymer films prepared by each approach release NO for variable periods of time (10-72 h), although they differ in the mechanism, location and amount of NO released. In vitro platelet adhesion studies demonstrate that the localized NO release may prove to be an effective strategy for improving blood compatibility of polymer materials for a wide range of medical devices.     

Antithrombogenic surfaces: characterization and bioactivity of surface immobilized PGE1-heparin conjugate

A covalently bonded conjugate of commercial grade heparin and prostaglandin E1 (PGE1) was synthesized to prevent both fibrin formation and platelet aggregation during thrombus formation. The PGE1-heparin conjugate was immobilized on an imidazole carbamate derivatized sepharose bead surface through hydrophilic spacer groups (diamino-terminated polyethylene oxides). One end of the spacer group was coupled to the derivatized surface through a urethane bond between the amine group of the spacer and the derivatized surface. The free amine group of the immobilized spacers was coupled to a carboxylic group of the PGE1-heparin conjugate through an amide bond. Bioactivity of the immobilized conjugate (heparin activity) was measured in terms of increased clotting times (thrombin time assay) and for the inactivation of Factor Xa. Bioactivity of the immobilized compound (PGE1 activity) was analyzed by platelet adhesion and platelet release reactions using C14-5-hydroxytryptamine (5-HT). The conjugate immobilized via the C2 spacer showed the highest incidence of platelet adhesion, 5-HT released and the lowest activity for coagulation factors. In contrast, the 1000 and 4000 immobilized systems showed a significant reduction in platelet activation, while having the greatest effect on coagulation factors. The results of these experiments imply that the immobilized conjugate is active in preventing both pathways of thrombus formation, and the efficacy is improved through the use of long-chain hydrophilic spacer groups.     

Diazeniumdiolate-doped poly(lactic-co-glycolic acid)-based nitric oxide releasing films as antibiofilm coatings

Nitric oxide (NO) releasing films with a bilayer configuration are fabricated by doping dibutyhexyldiamine diazeniumdiolate (DBHD/N(2)O(2)) in a poly(lactic-co-glycolic acid) (PLGA) layer and further encapsulating this base layer with a silicone rubber top coating. By incorporating pH sensitive dyes within the films, pH changes in the PLGA layer are visualized and correlated with the NO release profiles (flux vs. time). It is demonstrated that PLGA acts as both a promoter and controller of NO release from the coating by providing protons through its intrinsic acid residues (both end groups and monomeric acid impurities) and hydrolysis products (lactic acid and glycolic acid). Control of the pH changes within the PLGA layer can be achieved by adjusting the ratio of DBHD/N(2)O(2) and utilizing PLGAs with different hydrolysis rates. Coatings with a variety of NO release profiles are prepared with lifetimes of up to 15 d at room temperature (23?°C) and 10 d at 37?°C. When incubated in a CDC flow bioreactor for a one week period at RT or 37?°C, all the NO releasing films exhibit considerable antibiofilm properties against gram-positive Staphylococcus aureus and gram-negative Escherichia coli. In particular, compared to the silicone rubber surface alone, an NO releasing film with a base layer of 30?wt% DBHD/N(2)O(2) mixed with poly(lactic acid) exhibits an ～98.4% reduction in biofilm biomass of S.?aureus and ～99.9% reduction for E.?coli at 37?°C. The new diazeniumdiolate-doped PLGA-based NO releasing coatings are expected to be useful antibiofilm coatings for a variety of indwelling biomedical devices (e.g., catheters).     

In vitro cytotoxicity of nitric oxide-releasing sol-gel derived materials

The cytotoxicity of bare and PU-coated nitric oxide (NO)-releasing sol-gel derived materials (sol-gels) was investigated using L929 mouse fibroblasts in both direct and indirect contact models to differentiate between the biological impact of the sol-gel matrix and NO release. The flux of NO was varied up to 150 pmol cm(-2) s(-1) using N-(6-aminohexyl)-aminopropyltrimethoxysilane (balance iso-butyltrimethoxysilane) diazeniumdiolate (NO donor)-modified sol-gels. The addition of a polyurethane (PU) outer membrane greatly improved the stability of the sol-gel matrix without significantly suppressing the NO flux. Direct contact studies demonstrated a cytotoxic effect that was dependent on the aminosilane content of the sol-gel. The use of the thin PU overcoat eliminated this effect. A direct cytotoxicity dependence of NO release for L929 fibroblasts was discovered from indirect contact studies, where 24 h exposure to NO fluxes in excess of 50 pmol cm(-2) s(-1) was cytotoxic.     

Analysis of immobilized L-cysteine on polymers

Recently, we reported that L-cysteine attached to polymeric biomaterials, without prior nitrosation, enhances the hemocompatibility of biomaterials via exploiting endogenous nitric oxide (NO). As part of the polymer optimization process to further enhance platelet inhibition, a kinetic model is being developed to predict the release rate of NO. A key model parameter is the immobilized concentration of L-cysteine. This article demonstrates how several chemiluminescence-based assays, previously utilized for measuring thiols in solution, were successfully adapted to quantify immobilized L-cysteine. The assays showed that the immobilized L-cysteine on the modified PET sample is within the range of 4.1 to 6.5 nmol/cm(2). An advantage of using the more successful chemiluminescence-based assay is that it can accurately measure molar concentrations of any thiol-containing compound with a detection limit in the pmol range. The major disadvantage is that L-cysteine must first be broken off of the polymer and released into solution prior to measurement, therefore leaving the sample unable to be reused. Other thiol-measuring techniques, such as fluorescence microscopy and X-ray photoelectron spectroscopy (XPS), were used to provide qualitative and semiquantitative analysis to substantiate the polymer development.     

Coils and tubes releasing heparin. Studies on a new vascular graft prototype

Coiled metallic guidewires find widespread use, for instance in interventional cardiology. It is known that release of heparin from the surface of guidewires is advantageous to prevent formation of thrombotic emboli. New coiled tubular structures, having larger inner and outer diameter as compared to guidewires, are presented. In theory these tubes can be used as interposition vascular grafts. Ten coiled tubes with an internal diameter of 690 microm were made. Five different adherent polymeric coatings with increasing hydrophilicity were used. Five tubes contained heparin in the coating and the other five were unheparinised controls. The five tubes containing heparin were studied with respect to heparin release in vitro (amount released, kinetics), and immobilised heparin that is exposed at the surface. All tubes were studied with a direct cell contact assay using 3T3 mouse fibroblast cells, a dynamic thrombin generation test, and endothelial cell growth onto the coils. It was found that the heparinised tubes lead to very little thrombin formation. It is argued that this is due to heparin that is immobilised and exposed at the inner surface of such tubes. Furthermore the coils showed to be cytocompatible and endothelial cells adhere and proliferate well onto the coils. This concept is believed to hold promise for further development of small vascular grafts.     

Heparinized polyurethanes: in vitro and in vivo studies

Heparin immobilization chemistry using alkyl spacer arms was adapted to optimize yield on polyurethane (PU) surfaces. The resultant biological activity of immobilized heparin (HI) was examined in vitro and in vivo, and compared with a heparin releasing (HR) system. Immobilized heparin retained its ability to bind and inactivate thrombin and Factor Xa; nonspecific coagulation factor binding was insignificant. Such activity cannot be attributed to the leakage of improperly bound heparin. Immobilized heparin-polyurethane catheters implanted in canine femoral and jugular veins for 1 h periods exhibited significant reduction in thrombus formation compared with untreated PU contralateral controls. Polyurethane catheters coated with a 9% heparin dispersion in PU (HR) system provided even greater improvement in antithrombogenicity.     

Modeling of degradation and drug release from a biodegradable stent coating

Biodegradable polymeric coatings on cardiovascular stents can be used for local delivery of therapeutic agents to diseased coronary arteries after stenting procedures. This can minimize the occurrence of clinically adverse events such as restenosis after stent implantation. A validated mathematical model can be a very important tool in the design and development of such coatings for drug delivery. The model should incorporate the important physicochemical processes responsible for the polymer degradation and drug release. Such a model can be used to study the effect of different coating parameters and configurations on the degradation and the release of the drug from the coating. In this paper, a simultaneous transport-reaction model predicting the degradation and release of the drug Everolimus from a polylactic acid (PLA) based stent coating is presented. The model has been validated using in vitro testing data and was further used to evaluate the influence of various parameters such as partitioning coefficient of water, autocatalytic effect of the lactic acid and structural change of the matrix, on the PLA degradation and drug release. The model can be used as a tool for predicting drug delivery from other coating configurations designed using the same polymer-drug combination. In addition, this modeling methodology has broader applications and can be used to develop mathematical models for predicting the degradation and drug release kinetics for other polymeric drug delivery systems.     

Thromboresistance characterization of extruded nitric oxide-releasing silicone catheters

Intravascular catheters used in clinical practice can activate platelets, leading to thrombus formation and stagnation of blood flow. Nitric oxide (NO)-releasing polymers have been shown previously to reduce clot formation on a number of blood contacting devices. In this work, trilaminar NO-releasing silicone catheters were fabricated and tested for their thrombogenicity. All catheters had specifications of L = 6 cm, inner diameter = 21 gauge (0.0723 cm), outer diameter = 12 gauge (0.2052 cm), and NO-releasing layer thickness = 200 ± 11 μm. Control and NO-releasing catheters were characterized in vitro for their NO flux and NO release duration by gas phase chemiluminescence measurements. The catheters were then implanted in the right and left internal jugular veins of (N = 6 and average weight = 3 kg) adult male rabbits for 4 hours thrombogenicity testing. Platelet counts and function, methemoglobin (metHb), hemoglobin (Hb), and white cell counts and functional time (defined as patency time of catheter) were monitored as measured outcomes. Nitric oxide-releasing catheters (N = 6) maintained an average flux above (2 ± 0.5) × 10(-10) mol/min/cm for more than 24 hours, whereas controls showed no NO release. Methemoglobin, Hb, white cell, and platelet counts and platelet function at 4 hours were not significantly different from baseline (α = 0.05). However, clots on controls were visibly larger and prevented blood draws at a significantly (p < 0.05) earlier time (2.3 ± 0.7 hours) into the experiment, whereas all NO-releasing catheters survived the entire 4 hours test period. Results indicate that catheter NO flux levels attenuated thrombus formation in a short-term animal model.     

Hydroxyapatite/poly(epsilon-caprolactone) composite coatings on hydroxyapatite porous bone scaffold for drug delivery

Hydroxyapatite (HA) porous scaffold was coated with HA and polycaprolactone (PCL) composites, and antibiotic drug tetracycline hydrochloride was entrapped within the coating layer. The HA scaffold obtained by a polymeric reticulate method, possessed high porosity ( approximately 87%) and controlled pore size (150-200 microm). Such a well-developed porous structure facilitated usage in a drug delivery system due to its high surface area and blood circulation efficiency. The PCL polymer, as a coating component, was used to improve the brittleness and low strength of the HA scaffold, as well to effectively entrap the drug. To improve the osteoconductivity and bioactivity of the coating layer, HA powder was hybridized with PCL solution to make the HA-PCL composite coating. With alteration in the coating concentration and HA/PCL ratio, the morphology, mechanical properties, and biodegradation behavior were investigated. Increasing the concentration rendered the stems thicker and some pores to be clogged; as well increasing the HA/PCL ratio made the coating surface be rough due to the large amount of HA particles. However, for all concentrations and compositions, uniform coatings were formed, i.e., with the HA particles being dispersed homogeneously in the PCL sheet. With the composite coating, the mechanical properties, such as compressive strength and elastic modulus were improved by several orders of magnitude. These improvements were more significant with thicker coatings, while little difference was observed with the HA/PCL ratio. The in vitro biodegradation of the composite coatings in the phosphate buffered saline solution increased linearly with incubation time and the rate differed with the coating concentration and the HA/PCL ratio; the higher concentration and HA amount caused the increased biodegradation. At short period (<2 h), about 20-30% drug was released especially due to free drug at the coating surface. However, the release rate was sustained for prolonged periods and was highly dependent on the degree of coating dissolution, suggesting the possibility of a controlled drug release in the porous scaffold with HA+PCL coating.     

Chemical–Physical Characterization of Polyurethane Catheters Modified with a Novel Antithrombin-Heparin Covalent Complex

Detailed structural studies were made of polyurethane catheter surfaces modified with a covalent antithrombin-heparin (ATH) complex that has superior anticoagulant activity compared to unfractionated heparin. ATH was grafted onto polyurethane catheters by surface film preparation involving a three-step process: (1) activation of ATH through functionalized poly(ethylene glycol) (PEG), (2) base-coating treatment of the polyurethane surface and (3) final attachment of ATH onto the surface by free radical polymerization. With the application of base coating, composed of polyhydroxyethylmethacrylates and poly(ethylene oxide) (PEO), the coating process could easily be transferred to other biomaterials by adjusting the base-coating composition. Anti-factor Xa assays confirmed high anticoagulant activity of the ATH coatings. To determine structural aspects critical for biological function, the product was analyzed using differential scanning calorimetry and SDS-PAGE. Radiolabeled ATH was used to determine the graft density, homogeneity and stability of modified surfaces, as well as the competition of PEO-ATH migration to the surface with self-aggregation of the PEO-ATH molecules during the coating process. X-ray photoelectron spectroscopy was used to investigate the surface chemical composition before and after ATH application. Analysis showed that PEO-ATH was strongly surface-bound at a final density of 15–200 pmol/cm², depending on the incubation concentration.     

The effect of calcium ions on the activated partial thromboplastin time of heparinized plasma

The effect of calcium chloride (CaCl2) on the activated partial thromboplastin time (aPTT) of heparinized plasma was studied. The aPTT ratio (heparinized plasma:control plasma) increased as the CaCl2 concentration to recalcify the plasma was increased from 15 to 35 mmol/L CaCl2. Platelet-poor plasma from patients receiving intravenous heparin, and in vitro heparinized plasmas from either coumarinized patients or plasma depleted of the vitamin K-dependent factors, displayed the calcium-dependent increase in the aPTT ratio. The magnitude of the calcium-dependent change in the aPTT ratio was similar for the three partial thromboplastins studied. Heparinized blood collected in 3.2% and 3.8% citrate demonstrated the calcium-dependent increase in the aPTT ratio. The authors have also studied the effect of the divalent cations (Ca+2, Mg+2, Zn+2, and Sr+2) on the anti-Factor Xa activity of heparin to determine whether the calcium-dependent increase in the aPTT was due to an increase in the anti-Factor Xa activity. The anti-Factor Xa activity of heparin was measured using chromogenic substrate S-2251, purified Factor Xa, and excess antithrombin III. The anti-Factor Xa activity of heparinized plasma increased 2.4-2.8-fold as the divalent cation concentration was increased from 0-5 mmol/L. Similar results were obtained using purified bovine Factor Xa, antithrombin III, and heparin in the absence of plasma. These results suggest that divalent cations play an important role in modulating heparin's anticoagulant activity in vitro. In addition, the CaCl2 concentration used to recalcify plasma is an important variable that modifies heparin sensitivity of the aPTT. Furthermore, divalent cations play an important role in regulating the anti-Factor Xa activity of heparin in vitro.     

Initiated chemical vapor deposition of antimicrobial polymer coatings

The vapor phase deposition of polymeric antimicrobial coatings is reported. Initiated chemical vapor deposition (iCVD), a solventless low-temperature process, is used to form thin films of polymers on fragile substrates. For this work, finished nylon fabric is coated by iCVD with no affect on the color or feel of the fabric. Infrared characterization confirms the polymer structure. Coatings of poly(dimethylaminomethyl styrene) of up to 540 microg/cm2 were deposited on the fabric. The antimicrobial properties were tested using standard method ASTM E2149-01. A coating of 40 microg/cm2 of fabric was found to be very effective against gram-negative Escherichia coli, with over a 99.99%, or 4 log, kill in just 2 min continuing to over a 99.9999%, or 6 log, reduction in viable bacteria in 60 min. A coating of 120 microg/cm2 was most effective against the gram-positive Bacillus subtilis. Further tests confirmed that the iCVD polymer did not leach off the fabric.