Fig. 6.6 Cross-sectional microstructure of the Cu(0)-pHEMA hybrids (a) Hybrid-1-Cu05, and (b) Hybrid-4-Cu05, where microporous morphology is clearly shown as starting water concentration was increased.
6.5 Cu(Ⅱ) release Effect of Microstructure
The influence of microstructural variation of the hybrid on the resulting Cu release profile was evaluated using the hybrids with the addition of 0.5 wt% Cu, i.e., Hybrid-1-Cu05, Hybrid-3-Cu05 and Hybrid-4-Cu05, where accordingly higher porosity and larger pore size with increasing H2O/pHEMA ratio were observed as aforementioned. The cumulative release of Cu(Ⅱ) from the Cu(0)-pHEMA hybrids was measured and given directly in Fig.6.7, where a continuous release for a time duration of 240 hours is attained.
The parameters n of Hybrid-1-Cu05, Hybrid-3-Cu05 and Hybrid-4-Cu05 calculated from Eq. (2) are 0.602, 0.629, and 0.645 respectively from a highly-correlated linear regression analysis, indicating a non-Fickian diffusion mode. However, for those porous hybrids, e.g., Hybrid-3-Cu05 and Hybrid-4-Cu05, the release mechanism appears to be constant, compared with the dense one, i.e., Hybrid-1-Cu05. In other words, porosity or pore size, i.e., 3-10 um in diameter, evolved seems to have little or no effect on the diffusion mechanism of the hybrids, but the rate constant, k, which is increased with the increasing H2O/HEMA molar ratio from 0.0409 to 0.0747. The presence of porosity should have influenced on the release rate since the porous structure, i.e, Hybrid-3-Cu05 and Hybrid-4-Cu05, allows more surface area of the Cu particles to be exposed to the aqueous solution than that of dense structure, i.e., Hybrid-1-Cu05. Since the exponent n has a relatively constant value for these hybrids with various pore sizes, the value k can then be considered as an index of the
20μm
(b)
20μm (a)
estimated higher by 82.6% than that of the dense Hybrid-1-Cu05. Therefore, based on the structural evolution and a corresponding tunable release kinetic for the hybrids synthesized in current investigation, the Cu(0)-pHEMA hybrids with tunable technical merits with highly regulated elution of Cu can be well designed for biomedical uses.
Fig. 6.7 Cu(Ⅱ) release from the Cu(0)-pHEMA hybrids with different H2O/HEMA molar ratio. The release patterns showed a relatively slow and sustained kinetics with the values of k and n determined according to Eq. (2) for Mt/M∞
< 0.6 (n = 5).
Effect of Particle size and Concentration
Figure 6.8(a) shows the release behavior of cupric ion Cu(II) from Cu-loaded pHEMA hybrids. and composites prepared in this study, namely, the Cu(0)-pHEMA hybrids, i.e., with Cu size of 5~25 nm prepared in-situ. For comparison purpose, the Cu-pHEMA composites with commercial nano-particles (50nm, Sigma-Aldrich, Inc. USA) were prepared by using UV irradiation to aqueous solution containing the same composition as aforementioned Hybrid-1-Cu05, designated as Composite-1-Cu05. The Hybrid-4-Cu05 showed a apparently monotonical decrease with the release time, which is believed to be due to more porous pHEMA matrix as shown in Fig. 6.6(b). In contrast, for Hybrid-1-Cu05, the copper released relatively constantly, i.e., at a concentration of ~ 5.5 ppm over a regular time interval of 24 h, from the very beginning to a time duration of as long as 240 hours. As a larger particle size was used, i.e., Composite-1-Cu-05, although the composite also showed a monotonical decrease over a time period of first 96 hours, followed by a slow reduction up to 240 hours, the slower profile was observed for the Cu released from the
10 100 1000
1 R2 = 0.981
R2 = 0.989
Hybrid-4-Cu05 Hybrid-3-Cu05 Hybrid-1-Cu05
M t/M
Time (hrs) R2 = 0.972 8
composites, indicating a lower surface area exposed to the surrounding aqueous environment for the larger size of the Cu particles embedded in the pHEMA matrix.
Fig. 6.8 (a) Curves of Cu(Ⅱ) release rates of in-situ Cu(0)-pHEMA hybrids and Cu(0)-pHEMA composites in the PBS. (b) Dependence of n value of different copper content for Cu(Ⅱ) release from Cu(0)-pHEMA composites and in-situ Cu(0)-pHEMA hybrids.
Although no direct evidence for the mechanism of Cu(0) Æ Cu(II) can be provided in this study, it is generally recognized that an oxidation occurred on the surface of the Cu nanoparticles, following a dissolution of the Cu(II) into the medium. Therefore, the release kinetic is considered as an apparent kinetic of Cu(II) release. The diffusion mechanism of the Cu(II) from the hybrids can be characterized using Equation (2) [172-173]:
t ktn
MM =
∞
(2)
where Mt is the mass of Cu(Ⅱ) released at time t, M∞ the mass of released Cu(Ⅱ) at time of infinity, k is a proportional constant, and n is a characteristic exponent related to the mode of transport of the solute. This equation is applicable for Mt/M∞ < 0.6. Three modes of diffusion release mechanism were defined from various controlled release systems [174]. Fickian diffusion (n = 0.5), in which the rate of diffusion is much slower than the rate of swelling; in this case, the system is controlled by diffusion. The second mode is Case II transport (n = 1.0), where the diffusion process is much faster than the swelling process. The controlling step is the velocity of an advancing front, i.e., a swelling-controlled mode. The third is non-Fickian (anomalous) transport (0.5 < n < 1.0), which describes those cases where the diffusion and
50 100 150 200 250
For comparison, a series of Cu(0)-pHEMA hybrids with the same load of copper as that of the composites were prepared. The parameters n and k calculated from Eq. (2) are listed in Table 6.1. For the hybrids, the parameters n of Hybrid-1-Cu01, Hybrid-1-Cu05 and Hybrid-1-Cu10 are 0.629, 0.668, and 0.719 respectively, indicating the release of Cu(Ⅱ) from the Cu(0)-pHEMA hybrids is non-Fickian diffusion, and can be categorized as a combination of both diffusion and swelling controlled modes. In other words, structural evolution upon dilation or relaxation of the hybrid may play certain critical role in determining the resulting release kinetics for the hybrids. This is more pronounced since the Cu particles in the hybrids are the smallest size among other samples and a much higher dissolution rate is heavily expected compared with others. Under such conditions, the length of the pathway and tortuosity of the hybrid structure may play important roles in releasing rate. However, for the composites, namely, Composite-1-Cu01, Composite-1-Cu05 and Composite-1-Cu10, the values of n are 0.582, 0.595, and 0.636 respectively, smaller than the ones from the hybrids, indicating the release of Cu(Ⅱ) from the Cu-pHEMA composite also exhibited a non-Fickian type of diffusion that dominates the release behavior as that of the hybrids.
Table 6.1 The parameters n and k calculated from Eq. (2) of Cu(0)-pHEMA composites and hybrids.
k n r-square
Composite-1-Cu01 0.0055 0.582 0.985 Composite-1-Cu05 0.0074 0.595 0.982 Composite-1-Cu10 0.0078 0.636 0.972 Hybrid-1-Cu01 0.0018 0.629 0.982 Hybrid-1-Cu05 0.0099 0.668 0.986 Hybrid-1-Cu10 0.0116 0.719 0.969
However, the n value was found to increase linearly with the content of Cu in both the hybrid and composite samples, Fig 6.8(b), suggesting that the Cu release mechanism may apparently be a combination of both diffusion and swelling modes. This is because a certain extent of agglomeration of the Cu particles, for both the hybrids and composites, was observed when the concentration of the Cu was increased from 0.1% to 1.0%. Such a structural inhomogeneity may contribute to the resulting swelling mode of release kinetics.
Therefore, we believed that the linear change of the n along with Cu concentration given in
Fig. 6.8(b) indicates a possible transition of release mechanism from diffusion to swelling, resulting in an apparently non-Fickian mode of Cu release.
6.6 Cell behavior
The influence of Cu release and the corresponding concentration on cellular behavior is vital to practical applications. Figure 6.9 shows the growth behavior of the HUVEC with the Cu(II) concentration released from the Cu(0)-pHEMA hybrids for a time duration of 24h and 48h. Copper-induced growth of the endothelial cells was observed and shown to be significant for the hybrids with lower release profile, i.e., Hybrid-1-Cu05 and Hybrid-2-Cu05, in the range of 6~15 ppm daily. After 48-h incubation, a progressive increase of cell numbers which reached 121% of the control group was obtained. However, it seems not applicable for the hybrids with higher water content and porosity, such as Hybrid-3-Cu05 and Hybrid-4-Cu05, where the population of the endothelial cells is reduced to about 80% of the control, albeit not significant to the control group, indicating a higher concentration of Cu(II) released from especially the Hybrid-4-Cu05 prohibit the proliferation of the endothelial cells. Regardless of those biological concerns, in this short-term observation, the Cu-pHEMA hybrid prepared in this investigation has shown promising compatibility and proliferative activity to the endothelial cells. Although preliminary, this study envisions that the copper released form the hybrids shows short-term cytocompatibility at the concentration below certain level, i.e., 15 ppm. A stringent control release of the Cu from the hybrids is therefore critical for biomedical applications and through the manipulation of synthesis scheme, the hybrids capable of delivering optimal dose of Cu can be easily synthesized. However, the mechanism and signal transduction pathways for copper-induced endothelial cell proliferation are not clearly understood. A subsequent study is underway and will be reported shortly.
0
Cell number (% of control) Copper concentration (ppm)
Fig. 6.9 Effect of copper ion on HUVEC proliferation at various time periods.
Cell number was determined by AlamarBlue assay.
6.7 Summary
Novel nanocomposites with metallic Cu nanoparticles well-embedded within the pHEMA matrix, forming a well-defined nanostructure have been extensively characterized.
The interaction between Cu(II) with the hydroxyl groups within the pHEMA matrix was confirmed by the infrared spectral analysis and a corresponding improvement in the thermal stability of the Cu(0)-pHEMA hybrids. The molar ratio of H2O/HEMA in the starting synthesis plays a crucial role in the nanostructural evolution of the Cu nanoparticles, wherein a change from amorphous-like Cu or crystalline Cu, corresponding to a size ranging from ~ 5 nm to ~25 nm, has been observed when the ratio of H2O/HEMA is increased. In addition, the resulting hybrid evolved a microstructural development from dense to porous structure, causing a change in Cu release rate, while the release kinetic is considered to be a non-Fickian diffusion mode or plausibly, a combination of diffusion and swelling modes. Under well control of the hybrid composition, Cu release can be well regulated such as those of dense hybrids, i.e., Hybrid-1 and Hybrid-2, where a Cu(II)-induced proliferation of the HUVEC was observed for a 48-h incubation in vitro, and
agreed with literature report. In contrast, inhibition to the proliferation of the human smooth muscle cell line was observed for a longer incubation time. It may suggest a potential use of the Cu(0)-pHEMA nanocomposite for a number of biomedical applications, such as coating for cardiovascular stents, and will be reported separately.
Chapter 7
Metal/organic Hybrid – Cu/pHEMA –In-Situ Synthesis of Hybrid Nanocomposite with Highly-Order Arranged Amorphous Metallic Copper
Nanoparticle in Poly(2-hydroxyethyl methacrylate) and Its Potential for Blood-contact Uses
7.1 Introduction
This has long been highlighted in the area of blood-contacting devices or implants, where topological texture, charging and hydrophilic nature of the hybrid surface are of great concerns [179, 180], and attentions have also largely paid to the electrochemical behavior of the materials surface in order to inhibit the occurrence of thrombosis [181-183]. A general understanding, albeit not yet being officially standardized, for a material showing blood compatibility can be characterized in terms of, for instance, platelet adhesion, fibrinogen deposition, and clotting time. However, it seems that current existing blood-contacting devices in the market are not as good as a true hemocompatible one from the viewpoints of biology and biomaterials science [184]. Development of new biomaterials with improved blood compatibility has continuously attracted great attention, and recently, thromboresistant materials based on inorganic, metals and metal-organic compound have received most attention, which have known to provide more advantages over conventional polymeric materials which are not truly thrombosis-free surface but currently widely available in the market [185, 186]. In situ synthesis of hybrid materials has long been technically attractive for improved property, for instance blood compatibility, due to desirable electrochemical behavior, better physical uniformity and higher chemical homogeneity of both surface and bulk regions. Here, a novel in-situ synthesis method is reported where a hybrid system based on the use of 2-hydroxyethyl methacrylate, i.e.,HEMA, monomers that were photopolymerized in the presence of Cu2+ precursor was prepared through an in-situ synthesis, following an in-situ chemical reduction of the Cu2+
precursor to form resulting metallic Cu-containing hybrid. The hybrids were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and streaming potential measurements. Interaction between blood and hybrids were examined in terms of platelet adhesion test of human whole blood.