In Vitro Release Kinetics
The procedure for drug release tests has been demonstrated in an earlier study [136]. In brief, the test was performed in a vessel containing 2 compartment chambers of equal volume; 1 chamber (considered as the donor side) contained 90 ml of 2000 ppm vitamin B12
in PBS (pH 7.4) and the other chamber was loaded with only drug-free PBS (pH 7.4). Each chamber was mechanically stirred during the test. The concentration of vitamin B12 in the medium was measured by a UV-VIS spectrometer (SP-8001; Metertech Inc.) at 361 nm.
The permeability was calculated according to Eqn. (3-1) [136]:
V
where H is the partition coefficient, D is the effective diffusion coefficient, A is the effective area of the membrane, δ is the thickness of the membrane which was measured by digital thickness gauges (ASTM D926) with 5 parallel measurements for each sample, V is the solution volume (90 ml), C10 is the initial concentration (at t = 0) of the drug in the donor solution. C1 and C2 are the concentrations in the donor and receptor chambers, respectively, at a given time period (t = t) of diffusion. By plotting ln[C10/(C1 – C2)] versus time, the permeability [DH] can be obtained from the slope of the line. The apparent partition coefficient (H) of vitamin B12 for pHEMA, p(water/pHEMA), and SiO2/pHEMA composite membranes can be determined as follows [137]. The wet weight (W) of the membranes was recorded after immersing drug-free membranes (1 g) in the release medium (drug-free PBS,
pH 7.4) for 2 days. The wet membranes were then immersed in 10 ml of 2000 ppm vitamin B12-containing medium. The partition coefficient (H) is determined from the initial (C0) and equilibrium (Ce) concentrations of vitamin B12-containing mediums by Eqn. (3-2):
H = 10(C0 – Ce)/WCe. (3-2)
Platelet Adhesion Assessment
Fresh blood was drawn from a healthy adult volunteer. Care was taken to insure that it was free of aspirin and other drugs, such as anticoagulant, that could bias the results. Fresh blood and platelet adhesion tests were performed in vitro, to investigate the quantity of the adherent platelets on the Cu(0)-pHEMA hybrid. Cu(0)-pHEMA hybrid, i.e., 20C, with 1cm × 1cm in dimensions were immersed in 0.5ml platelet rich plasma (PRP) solution in the 24-well plate and then incubated at 37°C for 2 hr. After terminating the adhesion test by adding 0.5ml PBS into each well, samples were subsequently rinsed with a PBS buffer solution three times to remove the weakly adherent platelets. The firmly adhered blood components or platelets were fixed using 2.5% glutaraldehyde solution at 4 oC for 1 h. Samples with adherent blood components or platelets were then dehydrated in graded ethanol solutions (30%, 50%, 70%, 95%, and 100% for 15 min, respectively, at room temperature) and dried at 37°C for 24 h.
Then, the samples were characterized by field-emission scanning electron microscopy (FE-SEM, JAM-6700F) operated at 5 kV. Four SEM images were taken at a magnification of 500X for each sample and five parallel measurements were carried out for each hybrid. The numbers of platelets on the membrane surface were counted for each image and the counting area could be estimated by the scale bar. So the numbers and the volume fraction of platelet on the membrane surface were quantized. Volume fraction of thrombosis coverage on sample surface was counted on 20 fields chosen at random to obtain good statistic analysis.
Copper ion release test
A characteristic coloring is one of the most distinguishing features for cupric ion chelates and visual absorption spectra can be used to quantify the cupric ion content [138, 139].
Sodium diethyldithiocarbamate (copper reagent) was used to form a yellow chelate with the cupric ion. Spectrophotometric measurements were conducted using five concentrations of CuSO4 solution and adding copper reagent chelating agent. Absorbance (Abs) measurements were made at 1 nm intervals from 350 nm to 600 nm. The absorbance was measured using a UV-VIS spectrometer (001; Metertech I SP-8nc.) The calibration curve showed peak height (Abs) as a function of concentration of cupric ions. An absorption maximum can be observed at 450 nm and determinations were performed at this specific wavelength (450 nm). Absorbance measurements were calibrated using cupric sulphate solution and the copper reagent chelating agent. The release of cupric ion was determined by measuring the cupric ion content in the simulated uterine solutions, expressed as mg/L after 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours. Absorbance measurements were performed as follows: chelate solution was added to 10 mL of Cu(0)/pHEMA hybrid sample incubated solutions (pH=7.4 ) and the cupric ion content in the buffer solutions was then calculated from the measured absorbance and the calibration curve.
Cell culture
Endothelial cells (ECs, HUVEC, human umbilical vein endothelial cell, BCRC number:
H-UV001) were purchased from Food Industry Research and Development Institute (FIRDI), Taiwan. ECs were grown in 90% medium 199 with 25 U/ml heparin, and 30 μg/ml endothelial cell growth supplement (ECGS) adjusted to contain 1.5 g/l sodium bicarbonate and 10% fetal bovine serum. Smooth muscle cell (SMCs, T/G HA-VSMC smooth muscle cell, human normal aorta smooth muscle cell, BCRC number: 60293) were also purchased from FIRDI and were grown in culture medium containing 90% F-12K
medium with modifications-Ham’s F12K medium with 2mM L-glutamine adjusted to contain 1.5 g/l sodium bicarbonate supplemented with 10mM HEPES, 10mM TES, 0.05 mg/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite and 0.03 mg/ml ECGS and 10% fetal bovine serum. The cells were incubated in 75 cm2 culture flask (for ECs, the flask was coated with 1% gelatin (Sigma-Aldrich, Inc. USA ) before used) in 5% CO2 at 37 oC and culture medium renewal was carried out about every 2~3 days. After 5~10 days, the cells were removed from the 75 cm2 culture flask with trypsin (Gibco, Invitrogen, Taipei, Taiwan) into the 96-well culture plates with a cell concentration of 5× 103 cells/ml for 6 h and then incubated with pHEMA and Cu(0)-pHEMA hybrids for durations of 1 and 2 days.
Cell proliferation assay
The viability and proliferation of ECs or SMCs culture with pHEMA and Cu(0)-pHEMA hybrids at specific durations were determined by AlamarBlue™ assay (AbD Serotec Ltd, Oxford, UK). AlamarBlue™ assay is designed to quantitatively measure the proliferation of various human and animal cell lines. AlamarBlue™ assay was diluted in an amount equal to 10% of PBS volume. At the specified durations (1 and 2 days), pHEMA or Cu(0)-pHEMA hybrids and culture medium were removed from the 96-well culture plate, and 100μl of diluted AlamarBlue™ assay was added to each well and the plates were incubated at 37 oC under 5% CO2 for 4 h. The absorbance, or called optical density (OD), of AlamarBlue™ assay (color from blue to purple) was measured at 590 nm with an ELASA spectrophotometer. The proliferation of endothelial cells (HUVEC) and smooth muscle cells (T/G HA-VSMC) cultured in medium without the endothelial cell growth supplement (ECGS) was also tested in contrast. All experiments were repeated five times. Data were taken to be significant, when a p-value of 0.05 or less was obtained (showing a 95%
Zeta potential measurement
The surface charge (zeta potential) of the pHEMA and hybrid membranes were characterized using a streaming potential apparatus, which has been detailed elsewhere [140]. In brief, an electrolyte solution was prepared with a 10-3 M KCl solution (dissolved in ultrapure water) at pH 7.4 where the pH value was adjusted by the addition of 0.1M NaOH or 0.1M HCl solutions. In each experiment, the solution flow through the membrane module was supplied by peristaltic pump and the trans-membrane pressure was controlled between 0.2 ~ 1 bar, while the crossflow velocity of the solution was fixed at 0.1 m/s. For each sample, four values of pressure difference (ΔP) were measured and the data of streaming potential (ΔE) were recorded by a personal computer. The zeta potential ζ was calculated using the Helmholtz-Smoluchowski equation, eq. (3-3), where κ is the liquid conductivity, η is the liquid viscosity, εr is the liquid permittivity, εo is the permittivity of free space, ΔE is the streaming potential, and ΔP is the hydrodynamic pressure difference. The zeta potential ζ was calculated from the slope of ΔE/ΔP curve which having a R-square of the regression curve larger than 0.99, i.e. r2 > 0.99 for each sample.
P E
r Δ
= Δ ε0
ε
ζ κη (3-3)
Electrochemical tests
Electrochemical tests were performed using a standard three-electrode, temperature-controlled cell and a microprocessor controlled potentiostat (CH instruments, CHI, model 614A). An Ag/AgCl (in 3M NaCl) electrode was used as the reference and platinum plates were used as the working and counter electrode. Cyclic voltammetry (CV) analysis was carried out in 0.1M NaOH solution at room temperature from -700 to +700 mV, at a rate of 50 mV/sec versus an Ag/AgCl reference electrode. A three-electrode system was used throughout the study. Pt plates were used as the working and counter electrode. Cu/pHEMA film clipped tightly
between working and counter electrode and the surface of the Cu/pHEMA sample was stuck smoothly on each of the electrode. Analysis of cyclic voltammetry tracings and determination of the oxidation potential and the anodic current are reported. An alternating current (AC) impedance measurement technique was employed to investigate the electrochemical kinetics at the Cu/pHEMA- electrolyte interface. The measurement was performed at an open-circuit potential and the frequency was varied in the range of 105 Hz to 10-2 Hz with an imposed voltage of 5 mV AC (model 614A, CHI). These experiments allow the detection of the properties of the films when submerged in an electrolyte solution. Parameters such as capacitance and resistance are generally recorded. The time constant, τ, is calculated by multiplying the values of capacitance and electrical resistance obtained from the impedance measurements.