Improving the thromboresistivity of chemical sensors via nitric oxide release: fabrication and in vivo evaluation of NO-releasing oxygen-sensing catheters

The development and in vivo analytical performance of a nitric oxide (NO)-releasing amperometric oxygen sensor with greatly enhanced thromboresistivity are reported. Gas permeable coatings formulated with cross-linked silicone rubber (SR) containing NO-generating compounds (diazeniumdiolates) are shown to release NO for extended periods of time (> 20 h) while reducing platelet adhesion and activation. Oxygen-sensing catheters prepared by dip-coating the NO-releasing films over the outer SR tubes of the implantable devices display similar analytical response properties in vitro (sensitivity, selectivity, response times) when compared to analogous sensors prepared without the NO release coatings. Superior analytical accuracy (relative to blood PO2 values measured in vitro) and greatly reduced thrombus formation on the outer surface of the sensors are observed in vivo (in canine model) with the NO release PO2 sensors compared to control sensors (without NO release) implanted simultaneously within the same animals. Based on these preliminary studies, the use of NO release polymers to fabricate catheter-style chemical sensors may be a potential solution to lingering biocompatibility and concomitant performance problems encountered when attempting to employ such devices for continuous intravascular measurements of blood gases and electrolytes.     

Crosslinked duplex DNA nanogels that target specified proteins

Specific detection of protein biomarkers plays an important role in diagnostics and therapeutics. We have fabricated polymeric nanogels, which can specifically interact with the cancer biomarker thrombin to serve as a model. Two types of 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymers bearing a thrombin-binding oligonucleotide aptamer and its complementary chain were independently synthesized by redox-initiated radical polymerization. These MPC polymers associate in a complimentary fashion due to double strand formation of the oligonucleotides in aqueous media, leading to the spontaneous formation of spherical nanogels. Nanogel formation was confirmed by dynamic light scattering (DLS) and transmittance microscopy. The average size of nanogel particles was 124 ± 2 nm and the nanogels were mono-dispersed (polydispersity index 0.21). Functional intercalators could be stably incorporated into nanogels through the physical interaction between the intercalators and the oligonucleotides. The ethidium bromide (EtBr)-incorporating nanogels were used as detectors for thrombin. The fluorescence intensity of solutions containing the EtBr-incorporating nanogels was decreased with an increase in the concentration of thrombin. The transformation of quadruplex–thrombin structure from complementary double-stranded structures resulted in the decrease in fluorescence intensity. In contrast, the intensity did not change when the nanogels were incubated with albumin. Thrombin is only one such model used to demonstrate this technique; oligonucleotide aptamers can be freely designed to interact with versatile bio-substances. Therefore, aptamer-crosslinked nanogels can be appropriate nanomaterials for disease diagnosis and therapy.     

Preparation of water dispersible,fluorescent Ag–PAA–PVP hybrid nanogels and their optical properties

A simple method of synthesizing hybrid silver–polyacrylic acid–poly(N-vinylpyrrolidone) (Ag–PAA–PVP) nanogels was demonstrated through in situ reducing Ag⁺ inside PAA–PVP nanogels, which were formed by polymerization of acrylic acid in the PVP solution. Due to the ion exchange between Ag⁺ and acid protons of PAA, stable Ag⁺ clusters were formed inside the PAA–PVP nanogels, and hybrid nanogels were obtained by reducing Ag⁺ by ascorbic acid. Transmission electronic microscopic (TEM) images clearly showed the existence of silver nanoparticles inside the Ag–PAA–PVP nanogels. These hybrid nanogels showed typical surface plasma resonance absorption peak around 420 nm, and the size of the silver nanoparticles inside the Ag–PAA–PVP nanogels could be controlled from 9.5 ± 1.6 nm to 1.9 ± 0.4 nm by increasing the feeding amount of Ag⁺. In addition, these hybrid nanogels showed photoluminescent properties in fluorescent spectra. Considering the pH sensitive property of these hybrid nanogels, they will have potential application in drug delivery and biomedical imaging systems.     

Integrating blood cell mechanics,platelet adhesive dynamics and coagulation cascade for modelling thrombus formation in normal and diabetic blood

Normal haemostasis is an important physiological mechanism that prevents excessive bleeding during trauma, whereas the pathological thrombosis especially in diabetics leads to increased incidence of heart attacks and strokes as well as peripheral vascular events. In this work, we propose a new multiscale framework that integrates seamlessly four key components of blood clotting, namely transport of coagulation factors, coagulation kinetics, blood cell mechanics and platelet adhesive dynamics, to model the development of thrombi under physiological and pathological conditions. We implement this framework to simulate platelet adhesion due to the exposure of tissue factor in a three-dimensional microchannel. Our results show that our model can simulate thrombin-mediated platelet activation in the flowing blood, resulting in platelet adhesion to the injury site of the channel wall. Furthermore, we simulate platelet adhesion in diabetic blood, and our results show that both the pathological alterations in the biomechanics of blood cells and changes in the amount of coagulation factors contribute to the excessive platelet adhesion and aggregation in diabetic blood. Taken together, this new framework can be used to probe synergistic mechanisms of thrombus formation under physiological and pathological conditions, and open new directions in modelling complex biological problems that involve several multiscale processes.     

Short term evaluation of material blood compatibility using a microchannel array

New short-term evaluation of material blood compatibility was attempted using a microchannel array with human blood under a flow condition. The microchannel array chips were made of silicon, having 8,736 microchannels of 10 μm-wide, 30 μm-long, and 4.5 μm-deep on the average, as the models of capillary blood vessels. Titanium, chromium, albumin and collagen were coated onto the chips to examine the difference of material blood compatibility and the effect of protein adsorption on it. The time for the first 100 μl portion of whole blood to pass through the channels (blood pass-through time, BPT) was measured under a pressure difference of 20 cmH₂O. Simultaneously, the flow behavior of blood cells was observed by an optical microscope. The BPT tends to correlate well with the level of platelet adhesion. The highest BPT as well as platelet adhesion was observed on collagen, followed by titanium, chromium, silicon, and albumin. These results indicate that the BPT can detect the different levels of platelet adhesion and thrombus formation on microchannel surface and that the protein adsorption onto chip surface can influence BPT. We concluded that this method could be applied to evaluate initial blood compatibility of materials within several minutes in vitro.     

Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane with highly effective blood compatibility via atmospheric plasma-induced surface copolymerization

Development of nonfouling membranes to prevent nonspecific protein adsorption and platelet adhesion is critical for many biomedical applications. It is always a challenge to control the surface graft copolymerization of a highly polar monomer from the highly hydrophobic surface of a fluoropolymer membrane. In this work, the blood compatibility of poly(vinylidene fluoride) (PVDF) membranes with surface-grafted electrically neutral zwitterionic poly(sulfobetaine methacrylate) (PSBMA), from atmospheric plasma-induced surface copolymerization, was studied. The effect of surface composition and graft morphology, electrical neutrality, hydrophilicity and hydration capability on blood compatibility of the membranes were determined. Blood compatibility of the zwitterionic PVDF membranes was systematically evaluated by plasma protein adsorption, platelet adhesion, plasma-clotting time, and blood cell hemolysis. It was found that the nonfouling nature and hydration capability of grafted PSBMA polymers can be effectively controlled by regulating the grafting coverage and charge balance of the PSBMA layer on the PVDF membrane surface. Even a slight charge bias in the grafted zwitterionic PSBMA layer can induce electrostatic interactions between proteins and the membrane surfaces, leading to surface protein adsorption, platelet activation, plasma clotting and blood cell hemolysis. Thus, the optimized PSBMA surface graft layer in overall charge neutrality has a high hydration capability and the best antifouling, anticoagulant, and antihemolytic activities when comes into contact with human blood.     

MMA/MPEOMA copolymers as coating materials for improved blood compatibility: protein adsorption study

Surface-induced thrombosis remains one of the main problems in the development of blood-contacting devices. When a foreign surface comes in contact with blood, the initial blood response is adsorption of blood proteins, followed by platelet adhesion and activation, leading to thrombus formation. A particularly effective polymer for the prevention of protein adsorption and platelet adhesion appears to be polyethylene oxide (PEO). In this study, water-insoluble copolymers of methyl methacrylate (MMA) and methoxy PEO monomethacrylates (MPEOMA) with different PEO molecular weights (200, 400, and 1000) and monomer composition were synthesized and characterized by gel permeation chromatography and ¹H-nuclear magnetic resonance spectroscopy. The synthesized copolymers were coated on glass slides by a spin coating method to prepare PEO-rich surfaces as blood-compatible surfaces. The surface properties of the copolymers and their interaction with blood proteins (albumin, γ-globulin, fibrinogen, and plasma proteins) were investigated by the measurement of water contact angles and by electron spectroscopy for chemical analysis, respectively. It was observed that the protein adsorption on the copolymer surfaces decreased with increasing PEO molecular weight and MPEOMA content in the copolymers. The copolymers with long PEO chains in MPEOMA (MMA/MPEO₀₀₀MA copolymers) were effective in preventing protein adsorption, even though their MPEOMA content was less than the copolymers with shorter PEO chains. © 1999 Kluwer Academic Publishers     

Surface characteristics and blood compatibility of PVDF/PMMA membranes

Poly(vinylidene fluoride) (PVDF)/poly(methyl methacrylate) (PMMA) membranes have important applications as biomaterials. In this study, PVDF/PMMA with varying ratios is blown into membranes, the surface property and blood compatibility of which are thoroughly investigated. Membrane surface characteristics including composition and topography exert considerable influence on the blood compatibility. Introduction of PMMA disturbs PVDF crystallization, however, favors the β phase crystal formation. PVDF content, crystallization ability, and surface enrichment have decisive effects on the membrane surface composition. Meanwhile, with increased PMMA fraction, the membrane surface roughness is also increased, and subsequently results in decreased hemocompatibility. While the membranes with PMMA content lower than 30 wt% show good blood compatibility, those with higher PMMA fraction exhibit obvious platelet adhesion to the surface. Thermal annealing promotes the formation of β phase PVDF and generates much smoother surface, thus endowing the membranes with greatly enhanced blood compatibility. These results show the prospect for optimization of processability, surface property, and blood compatibility of PVDF/PMMA membranes through facile modulation of PMMA content and fabrication process.     

Improved thrombogenicity on oxygen etched Ti6Al4V surfaces

Thrombus formation on blood contacting biomaterials continues to be a key factor in initiating a critical mode of failure in implantable devices, requiring immediate attention. In the interest of evaluating a solution for one of the most widely used biomaterials, titanium and its alloys, this study focuses on the use of a novel surface oxidation treatment to improve the blood compatibility. This study examines the possibility of using oblique angle ion etching to produce a high quality oxide layer that enhances blood compatibility on medical grade titanium alloy Ti6Al4V. An X-ray photoelectron spectroscopy (XPS) analysis of these oxygen-rich surfaces confirmed the presence of TiO₂ peaks and also indicated increased surface oxidation as well as a reduction in surface defects. After 2 h of contact with whole human plasma, the oxygen etched substrates demonstrated a reduction in both platelet adhesion and activation as compared to bare titanium substrates. The whole blood clotting behavior was evaluated for up to 45 min, showing a significant decrease in clot formation on oxygen etched substrates. Finally, a bicinchoninic acid (BCA) total protein assay and XPS were used to evaluate the degree of key blood serum protein (fibrinogen, albumin, immunoglobulin G) adsorption on the substrates. The results showed similar protein levels for both the oxygen etched and control substrates. These results indicate that oblique angle oxygen etching may be a promising method to increase the thrombogenicity of Ti6Al4V.     

Copper-Based Metal-Organic Framework Induces NO Generation for Synergistic Tumor Therapy and Antimetastasis Activity

The interaction between platelets and circulating tumor cells (CTCs) contributes to distal tumor metastasis by protecting CTCs from immunological assault and shear stress, which can be disrupted by nitric oxide (NO) through inhibiting platelet-mediated adhesion. To eradicate primitive tumors and inhibit CTC-based pulmonary metastasis, a novel biomimetic nanomedicine (mCuMNO) is designed by encapsulating Cu⁺-responsive S-nitrosoglutathione as a NO donor into a copper-based metal-organic framework (CuM). This work discovers that mCuMNO can target tumor regions and deplete local glutathione (GSH) to reduce Cu²⁺ to Cu⁺, followed by triggering NO release and hydroxyl radicals ( · OH) production, thereby interrupting platelet/CTC interplay and contributing to chemodynamic therapy. Detailed studies demonstrate that mCuMNO exhibits high efficiency and safety in tumor therapy and antimetastasis activity, sheding new light on the development of CuM-based tumor synthetic therapy.     

Intramolecularly Protein‐Crosslinked DNA Gels: New Biohybrid Nanomaterials with Controllable Size and Catalytic Activity

DNA micro‐ and nanogels—small‐sized hydrogels made of a crosslinked DNA backbone—constitute new promising materials, but their functions have mainly been limited to those brought by DNA. Here a new way is described to prepare sub‐micrometer‐sized DNA gels of controllable crosslinking density that are able to embed novel functions, such as an enzymatic activity. It consists of using proteins, instead of traditional base‐pairing assembly or covalent approaches, to form crosslinks inside individual DNA molecules, resulting in structures referred to as intramolecularly protein‐crosslinked DNA gels (IPDGs). It is first shown that the addition of streptavidin to biotinylated T4DNA results in the successful formation of thermally stable IPDGs with a controllable crosslinking density, forming structures ranging from elongated to raspberry‐shaped and pearl‐necklace‐like morphologies. Using reversible DNA condensation strategies, this paper shows that the gels can be reversibly actuated at a low crosslinking density, or further stabilized when they are highly crosslinked. Finally, by using streptavidin–protein conjugates, IPDGs with various enzymes are successfully functionalized. It is demonstrated that the enzymes keep their catalytic activity upon their incorporation into the gels, opening perspectives ranging from biotechnologies (e.g., enzyme manipulation) to nanomedicine (e.g., vectorization).     

In vivo biocompatibility and analytical performance of intravascular amperometric oxygen sensors prepared with improved nitric oxide-releasing silicone rubber coating

The in vivo biocompatibility and analytical performance of amperometric oxygen-sensing catheters prepared with a new type of nitric oxide (NO)-releasing silicone rubber polymer (DACA/N2O2 SR) is reported. The NO-release silicone rubber coating contains diazeniumdiolated secondary amine sites covalently anchored to a dimethylsiloxane matrix. Narrow diameter (0.9 mm, o.d.) silicone rubber tubing coated with this polymer can be employed to construct functional oxygen-sensing catheters that release NO continuously at levels > 1 x 10(-10) mol/cm2-min for more than 20 h. In vivo evaluation of such sensors within the carotid and femoral arteries of swine over a 16-h time period demonstrates that sensors prepared with the new NO-release coating exhibit no significant platelet adhesion or thrombus formation, but control sensors (non-NO release) implanted within the same animals do show a high propensity for cell adhesion and bulk clot formation. Furthermore, the in vivo analytical data provided by sensors fabricated with NO-release coatings (N = 9) are shown to be statistically equivalent to PO2 levels measured in vitro on discrete samples of blood. Control sensors (N = 9) placed within the same animals yield average PO2 values that are statistically different (p < or = 0.05) (lower) from both the levels measured on discrete samples and those provided by the NO-release sensors over a 16-h in vivo monitoring period.     

Biocompatibility studies of polyacrylonitrile membranes modified with carboxylated polyetherimide

Poly (ether-imide) (PEI) was carboxylated and used as the hydrophilic modification agent for the preparation of polyacrylonitrile (PAN) membranes. Membranes were prepared with different blend compositions of PAN and CPEI by diffusion induced precipitation. The modified membranes were characterized by thermo gravimetric analysis (TGA), mechanical analysis, scanning electron microscopy (SEM) and contact angle measurement to understand the influence of CPEI on the properties of the membranes. The biocompatibility studies exhibited reduced plasma protein adsorption, platelet adhesion and thrombus formation on the modified membrane surface. The complete blood count (CBC) results of CPEI incorporated membranes showed stable CBC values and significant decrease in the complement activation were also observed. In addition to good cytocompatibility, monocytes cultured on these modified membranes exhibited improved functional profiles in 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Thus it could be concluded that PAN/CPEI membranes with excellent biocompatibility can be useful for hemodialysis.     

CD41 Western blotting: a new method to detect platelet adhesion to artificial surfaces used in extracorporeal circulation procedures

Cardiopulmonary bypass (CPB) surgery is associated with platelet activation and reduced platelet counts due to artificial surface activation of blood elements and non-physiological flow-patterns. As shown in former studies, coating of medical devices can improve hemocompatibility in extracorporeal circulation systems. In this study, we demonstrate a new method to determine platelet adhesion on 18 coated and non-coated membrane oxygenators in a simulated CPB model with CD41 Western blot. Platelet loss and the release of β-TG (platelet activation marker) were determined during a 120 min recirculation phase. At the end of the run the membrane oxygenators (with tubing system) were rinsed and the amount of adsorbed proteins on the surface was analyzed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting technique. Uncoated devices showed significantly higher concentrations of CD41 and of fibrinogen adsorption compared to the coated membrane oxygenators. These results correspond with the release of β-TG and platelet loss indicating less platelet adhesion on the coated oxygenators compared with the uncoated group. This new method may be useful in choosing less platelet activating materials for all kind of blood contacting devices to improve thrombogenicity including platelet functionality.     

Glow discharge plasma-induced immobilization of heparin and insulin on polyethylene terephthalate film surfaces enhances anti-thrombogenic properties

Polyethylene terephthalate (PET) films were treated with DC glow discharge plasma followed by graft copolymerization with acrylic acid (AA) and polyethylene glycol (PEG). The obtained PET–PEG was coupled to heparin or insulin molecules. The surfaces were then characterized by contact angle measurements, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The surface energies of the modified PET films were estimated using contact angle measurements, and the changes in crystallinity of the plasma-modified PET film surfaces were investigated by X-ray diffraction (XRD) analysis. The blood compatibilities of the surface-modified PETs were examined by in vitro thrombus formation, whole blood clotting time, platelet contact and protein adsorption experiments. The results revealed that the contact angle value decreased and that the interfacial tension between the modified PET films and blood protein was drastically diminished compared to unmodified PET film. The XPS results showed that the PET–AA surface containing carboxylic acid and the immobilized PET surface containing both carboxylic acid and amino groups exhibited a hydrophilic character, and AFM results showed marked morphological changes after grafting of AA, PEG and biomolecule immobilization. Heparin and insulin-coupled PET surfaces exhibited much less platelet adhesion and protein adsorption than the other surface-modified PET film surfaces.     

In vitro blood compatibility of poly (hydroxybutyrate-co-hydroxyhexanoate) and the influence of surface modification by alkali treatment

In vitro blood compatibility of poly (hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) was evaluated in comparison with poly (L-lactic acid) (PLLA) by a haemolysis assay, in vitro platelet adhesion test and coagulation measurements including plasma recalcification time (PRT), plasma prothrombin time (PT) and kinetic clotting time. The results showed that PHBHHx exhibited better blood compatibility than PLLA. Furthermore, PHBHHx film was modified by NaOH treatment to improve the surface hydrophilic property and the influence of the surface modification on the blood compatibility was investigated. Surface properties including hydrophilic property, surface appearance and functional groups were characterized by water contact angle measurement, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The results showed that the hydrophilic property of PHBHHx film was obviously improved by the NaOH treatment. It was also shown that the NaOH treatment could significantly enhance the blood compatibility of PHBHHx by prolonging PRT, PT, and kinetic clotting time and decreasing platelet activation. It is thought that the improvement in the hydrophilic property mainly contributes to the enhancement of blood compatibility.     

Detection of micro- and nano-sized biocompatible particles in the blood

The research deals with new scanning electron microscopic evaluations of the interface between blood and explanted temporary vena cava filters from patients affected by blood disorders. The biological tissues adherent to the filter and the small thrombi formed in vivo were detached from the metallic structure of the device, fixed, dehydrated and prepared for the histological and the electron microscopy. The analyses showed that both samples (thrombus and newly formed tissue) contained foreign, in some cases nano-sized, bodies. The chemistry of these particles was different and varied, and unusual compounds containing non-biocompatible elements like bismuth, lead, wolfram, tungsten were also detected. The interaction between these debris travelling in the blood stream and the blood itself leads to suspect that the formation of the thrombus can originate from these inorganic and inert foreign bodies that act as triggering agent of the blood coagulation.     

Anti-Platelet Effect Induced by Iron Oxide Nanoparticles: Correlation with Conformational Change in Fibrinogen

Iron oxide nanoparticles are developed for various biomedical applications, however, there is limited understanding regarding their effects and toxicity on blood components. The particles traveling in circulation inevitably interact with blood cells and plasma proteins and may interfere with hemostasis. Specifically, this study focuses on the influence of superparamagnetic iron oxide nanoparticles (SPIONs) coated with a biocompatible polymer, polyvinyl alcohol (PVA), on platelet function. Here, engineered SPIONs that are functionalized with various PVA coatings to provide these particles with different surface charges and polymer packing are described. These formulations are assessed for any interference with human platelet functions and coagulation, ex vivo. Positively charged SPIONs induce a significant change in platelet GPIIb-IIIa conformation, indicative of platelet activation at the dose of 500 µg mL⁻¹. Remarkably, engineered PVA(polyvinyl alcohol)-SPIONs all display a robust dose-dependent anti-platelet effect on platelet aggregation, regardless of the PVA charge and molecular weight. After assessing hypotheses involving SPION-induced steric hindrance in platelet–platelet bridging, as well as protein corona involvement in the antiplatelet effect, the study concludes that the presence of PVA-SPIONs induces fibrinogen conformational change, which correlates with the observed dose-dependent anti-platelet effect.     

Fluorescence determination of nitric oxide production in stimulated and activated platelets

Nitric oxide (NO) is quantitatively determined in platelets prior to, and after, stimulation with adenosine triphosphate (ATP) or activation with adenosine diphosphate (ADP). Platelets obtained from the whole blood of rabbits were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent NO production was measured as a fluorescent benzotriazole. Experiments were performed to determine the effect of probe concentration and probe incubation time in the platelets prior to measurement of the fluorescence. This information, combined with the method of multiple standard additions, was then employed to determine the moles of intracellular NO in the platelets (2.7 +/- 0.3) x 10(-16) mol of NO/platelet and the basal level of extracellular NO in the platelet sample (9.9 +/- 2.2) x 10(-18) mol of NO/platelet. Moreover, this method was used to quantitatively determine the amount of NO released from platelets whose NO production was stimulated with ATP (a nitric oxide synthase stimulus) or ADP, a substance known to result in NO production through platelet aggregation. When stimulated with ATP, the NO released from the platelets was determined to be (2.0 +/- 0.1) x 10(-17) mol of NO/platelet. When activated with ADP, the platelets released (2.8 +/- 0.3) x 10(-17) mol of NO/platelet. The difference between the extracellular basal levels of NO and that after stimulation with either ATP or ADP is in agreement with current estimates of NO release from platelets. Therefore, we conclude that a fluorescence determination of NO using the DAF family of probes, in combination with the method of multiple standard additions, can be employed to quantitatively determine the basal levels of NO in platelets, as well as the amount of NO released from stimulated and/or activated platelets.     

A multiscale model of thrombus development

A two-dimensional multiscale model is introduced for studying formation of a thrombus (clot) in a blood vessel. It involves components for modelling viscous, incompressible blood plasma; non-activated and activated platelets; blood cells; activating chemicals; fibrinogen; and vessel walls and their interactions. The macroscale dynamics of the blood flow is described by the continuum Navier-Stokes equations. The microscale interactions between the activated platelets, the platelets and fibrinogen and the platelets and vessel wall are described through an extended stochastic discrete cellular Potts model. The model is tested for robustness with respect to fluctuations of basic parameters. Simulation results demonstrate the development of an inhomogeneous internal structure of the thrombus, which is confirmed by the preliminary experimental data. We also make predictions about different stages in thrombus development, which can be tested experimentally and suggest specific experiments. Lastly, we demonstrate that the dependence of the thrombus size on the blood flow rate in simulations is close to the one observed experimentally.