The coagulation of blood consists of a cascade of reactions dividing into two pathways: the extrinsic and intrinsic pathways and platelets can accelerate thrombosis through secretion of bulk phase agonists, fibrinogen-mediated platelet–platelet aggregation, and by the acceleration of thrombin production [190]. Therefore, the extent of platelet adhesion to biomaterials is often used as an index of blood compatibility [191]. In this study, platelet adhesion and platelet activation on the surface of the Cu(0)-pHEMA hybrid was one of the main focuses in order to evaluate the hemocompatible properties of the hybrid. Fig. 7.5 shows that a trace amount of platelets adhered on the surface of the Cu(0)-pHEMA hybrid after 2 h immersion with human whole blood, in comparison with the PSF (polysulfonate, as a referenced materials) and pure pHEMA, a controlled group. The PSF showed a considerable amount of platelet adhesion, and pHEMA showed some extent of platelet adhesion, which are agreed with the observations reported in the literature [192, 193]. In contract to those two
0 50 100 150 200 250 300
Number of platelets adhered (20um x 20um)
PSF pHEM A Hybrid
well-recognized candidates, the amorphous metallic Cu(0)-pHEMA hybrid exhibits much improved anti-platelet adhesion behavior, where as small as 30% of the adhesion amount of platelet compared to that of neat pHEMA can be observed for the hybrids, and turned to be more significant in comparing to that of PSF, i.e., improved by about 92%.
Fig. 7.5 The blood platelet adhesion test showed that the Cu(0)-pHEMA hybrid demonstrated an improved anti-platelet adhesion behavior by ~70% and 92% compared to that of the neat pHEMA and PSF, respectively. (The scale bar of SEM image is 10μm.)
Although the exchange of electrons from blood proteins to the surface of medical devices has been considered to induce blood coagulation [179], the actual mechanism(s) of adhesion behavior of platelets from whole blood to the hybrid surface is not clearly understood at present. In spite of the ambiguity, it is reasonably trusted that the Cu nanoparticles in the pHEMA may trigger an electron-transferring process from the amorphous Cu(0) nanoparticles to blood-clotting proteins. In the meantime, the negatively-charged hybrid surface is expected
this will surely minimize or eliminate protein adsorption/denaturation as a result of electrostatic repulsion and prevent electron transferred from the protein to the negatively-charged hybrid surface. Either the former or latter mechanism or a combination can be effectively inhibiting undesirable platelet adhesion.
It seems able to conclude that the metallic Cu nanoparticle-pHEMA hybrids reported in this communication exhibited outstanding anti-platelet adhesion property compared to neat pHEMA and a widely-used biocompatible material, PSF. The highly-ordered nanostructure offers an unprecedented physical and chemical uniformity of the hybrids, together with its highly negatively-charged property, permitted the hybrids to behave as an outstanding anti-blood-clotting material. The electron-giving potential of the hybrid, as revealed in an earlier electrochemical test [179], together with the XPS spectral variation in Fig. 7.2, also reinforced the thromboresistant character of the hybrids. A further study on the blood compatibility in terms of Cu-catalyzed nitric oxide generation for the hybrids is under investigation through the use of electrochemical method and will be reported shortly.
7.6 Summary
The amorphous metallic Cu nanoparticles with an orderly packing configuration within the pHEMA matrix, forming a well-defined hybrid structure have been successfully synthesized.
The oxidation of Cu nanoparticles was confirmed by the XPS analysis. A relatively uniform copper nanoparticles about 2~5 nm in diameter and a relatively constant particle-to-particle distance of 8~10 nm were observed in Cu(0)-pHEMA hybrid structure, suggesting an unprecedented chemical and physical homogeneity can be achieved. The negative surface charge of the Cu(0)-pHEMA hybrid were confirmed by surface streaming potential and is expected to play important role in reducing the absorption of platelets. Thus, those experimental results demonstrate that the Cu(0)-pHEMA hybrid is capable of offering a promising characteristic for thromboresistant applications.
Chapter 8
Metal/organic Hybrid – Cu(0)-pHEMA - Electrochemical behavior of Cu(0)-pHEMA hybrid
8.1 Introduction
Over the last few years, much effort has been directed into developing new materials with specific electrochemical properties that could be used in biosensors, artificial muscles, and drug delivery system. The electric current of redox- reaction of specific enzyme and protein in biological substance could be used as an environmental signal to induce responses of designed action such as in-situ drug release or on–off switching from the implant device. On the other hand, the electrochemical properties of the implant material will also effect on the bio-reaction of the cell and implant materials [194]. For many biomaterials, interfacial charge on polymeric materials was claimed to be relevant for the success of medical devices according to different considerations [195]. Therefore, immobilization of ions, particles, and complex of metals in the matrix of polymeric for varying the electrochemical properties of the intrinsic polymer open up the way for designing the catalysts combining redox ability [196]
and provide a variable mechanism of activity in many physiological processes [197].
In this study, copper-containing pHEMA hybrids, Cu(0)-pHEMA, were developed for potential uses in blood-contact applications. An in-situ polymerization process was developed to promote high-affinity nucleation and growth of nano copper particles in the polymer hydrogel which provides an efficient approach toward inorganic/organic nano-hybrids with high-uniformity. These hybrids with different compositions could be produced and characterized in terms of their structure and electrochemical properties of the Cu(0)-pHEMA hybrid. In this chapter, an experimental study of different electro-chemical and physical aspects of electrochemical sensitive Cu(0)-pHEMA hybrid was presented. The
(AC) impedance measurements. The generation of nitric oxide in aqueous by Cu(0)-pHEMA hybrid was also tested in this study. The nano-sized Cu metal or metal oxide particles could disperse uniformly in polymer matrix and react with ionic group in the aqueous solution by the charge transfer from the interfacial surface of nano copper particles.
8.2 Fabrication of Cu-pHEMA Hybrid
The Cu-pHEMA hybrid was prepared by employing UV irradiation to an aqueous solution containing 2.5 grams of HEMA monomer, 0.1 ml of EDGMA as cross linker, 0.03 g BME as initiator, 0.1g SiO2 suspension, 0.5 grams H2O as solvent, and 0.1, 0.3, and 1 grams of 0.5 M CuSO4 solution with 0.1 wt% PVP for different hybrids which were named Hybrid-01, Hybrid-05, and Hybrid-10, respectively. The mixture was vigorously mixed during the course of reaction. To remove the dissolved oxygen from precursors, N2 was purged for approximately 1 min. Then, the mixture were transferred to a sealed transparent plastic holder of 50 mm × 50 mm × 0.5 mm in dimensions and UV irradiated at 365 nm for 2 h and the Cu(II)-pHEMA hybrid were produced. The Cu(II)-pHEMA hybrid were rinsed by distilled water to remove the un-reacted species (e.g., cross-linker, monomer, etc.) and then subjecting to in-situ chemical reduction by immersion the as-synthesized Cu(II)-pHEMA hybrid into 50 ml of 0.5 M hydrazine solution at 40oC for 24 h, to form a final metallic Cu(0)-pHEMA hybrid.
Cyclic voltammetry (CV)
CV analysis was carried out using the CH instruments potentiostat model 614A. CV scans of Cu(0)-pHEMA hybrids were in aerated Phosphate-buffered saline (PBS) at room temperature from -900 to -300 mV, at a rate of 20 mV/sec versus a Ag/AgCl reference electrode. A three-electrode system was used throughout the study. The working electrode was platinum plate covered with Cu(0)-pHEMA hybrid. Platinum wire served as the counter
electrode in all experiments. Analysis of CV tracings and determination of the oxidation potential and the anodic current are reported.
Alternating current (AC) impedance
An AC impedance measurement technique was employed to investigate the electrochemical kinetics at the semiconductor-electrolyte interface. The measurement was performed at an open-circuit potential and the frequency was varied in the range of 105 Hz to 10-1 Hz with an imposed voltage of 5 mV AC (CHI, model 614A). These experiments allow the detection of the properties of the films when submerged in an Phosphate-buffered saline (PBS) solution. Since the thin surface film acts as a capacitor when the semiconductor device makes contact with physiological fluids or blood, the time it takes for the reaction to take place is an important characteristic of each type of film. The time constant, τ, is calculated by multiplying of the values of capacitance and resistance obtained from the impedance measurements.
Nitric oxide generation in vitro
The NO-generating ability of Cu(0)-pHEMA hybrid was examined before and after the in vitro studies using a CH instruments potentiostat and nitric oxide detector (Mini Warn, KAOTEN science CO., LTD). Nitrosoglutathione (GSNO) was prepared by the reaction of equal-molar glutathione (GSH) and NaNO2 in 0.06M H2SO4. To n glass reaction cell containing 50mL PBS (138mM NaCl, 2.7mM KCl and 10mM sodium phosphate, pH 7.4) was added 1 μM GSNO, 30 μM GSH and 5 μM EDTA (to chelate metal ion contaminates that might otherwise decompose GSNO). The solution was bathed at 37 oC and continuously bubbled with N2. Any NO produced in the test solution was purged from the buffer, carried by the N2 gas into the nitric oxide detector chamber and monitored in real-time. The different GSNO concentration was controlled by adding 0.05M GSNO solution into reaction cell. The working potential is -0.8 mV versus a Ag/AgCl reference electrode.