Agarose (Type IX-A, Sigma), hydrochloric acid (HClO4, Aldrich), human umbilical vascular endothelial cells (HUVECs, Cambrex), endothelial cell medium-ECM (Cambrex), and MTS reagent (Promega) were used as received; however, pyrrole (Sigma) monomer was distilled and stored at -20 ℃ under argon until use. Indium tin oxide (ITO) conductive borosilicate glass slides with typical resistance of 30-60 Ω (Delta Technologies, Ltd.) were sequentially cleaned in acetone, methanol, isopropyl alcohol, and distilled deionized (DDI) water for 5 min each with ultrasonication. Slides were then dipped in a 1.0 N NaOH solution for 30 min, washed with copious amounts of DDI water, and dried under vacuum.
Preparation of Porous-structured PPy Scaffolds.
schematically depicts the hydrogel template procedure used for the preparation of porous-structured PPy films.
Pyrrole precursor solution was prepared by adding HClO
(1.0 M) to pyrrole (0.5 M) aqueous solution in 2:1 molar ratio and the solution was then deoxygenated at ambient temperature (~20 ℃) with argon (Ar) for 10 min to prevent oxidation of the monomers. While agarose was added to DDI water at 20 mg/mL, heated to over 90 ℃ until completely dissolved, and allowed to cool to 50-55 ℃. 500 μL of the agarose solution was then cast onto a 2.54 × 2.54 cm
ITO glass slide which was pre-warmed to 50-55 ℃ and allowed to cool to ambient temperature to form gel.
The obtained agarose gel served as templates for subsequent fabrication of porous-structured PPy.
The agarose gel on ITO slides was subsequently dipped into the pyrrole precursor solution for 24 h so as to soak through the entire gel and was saturated with the same concentration of the outer pyrrole solution. Electropolymerization of the pyrrole through the interstitial voids of the agarose gel template was carried out using a potentiostat/galvanostat (KST-P1, KOSENTECH) with a three-electrode electrochemical set up. The agarose gel template on ITO slide was used as the working electrode, a platinum mesh was served as a counter electrode, and a saturated calomel electrode served as a reference electrode. PPy films were deposited at an offset voltage of 720 mV in the pyrrole precursor solution. The film thickness was monitored and controlled by integrating the passage of current.
To remove the template, the PPy-agarose matrix on the ITO was then immersed into DDI water, which was heated to over 90 ℃ for overnight to selectively dissolve agarose out from the matrix. The resulting porous-structured PPy films were finally rinsed with copious amount of hot DDI water and dried at ambient temperature.
Chemical composition of the polymer surfaces was determined using a Physical Electronics (PHI) 5700 XPS equipped with an Al monochromatic source (Al KR energy of 1486.6 eV) and a hemispherical analyzer. The energy resolution was 1.0 eV for survey spectra and 0.1 eV for HR spectra. Binding energies were calibrated by setting the C-
component in the C(1s) envelope at 284.6 eV. The doping level was determined from the Cl/N ratio of HR Cl(2p) and N(1s) core-level XPS spectra. Conductivity at ambient temperature was measured with a conventional four-point probe resistivity apparatus placed on the polymer films. Film thickness was measured using a profilometer (Alpha Step 200, Tencor Instruments) and surface morphology was examined using scanning electron microscopy (SEM) (S-4000, Hitachi).
Cell Adhesion and Viability.
Cell adhesion studies were performed to validate the improved bioactivity of the porous- structured PPy. HUVECs were seeded in 1.5 cm
wells with 1 mL of ECM media at a density of 30 000 cells/cm
and cultured for 1 h in the absence of serum on the porous-structured films and a negative control conventional PPy. The colorimetric MTS cell proliferation assay was performed to quantify the extent of cell adhesion. Following 1 h of incubation in serum-free media, surfaces were rinsed three times by gently shearing 1 mL of 10 mM PBS over the film surface to remove unattached and loosely attached cells. PBS was aspirated, and new medium containing 2% fetal bovine serum (FBS) and 20% MTS reagent was added to each well and allowed to incubate at 37 ℃ for 1, 2, and 3 h time points. Cell viability was evaluated by removing 100 μL of media from the wells and measuring the absorbance of media at 490 nm using a microplate reader (Synergy
HT, BioTek). At least three samples were averaged to calculate each time point.
The research was supported by a grant from the Academic Research Program of Korea National University of Transportation in 2013.
Nanostructured Conductive Polymers
Chichester, U. K.
Andrews A. T.
Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications
Oxford, U. K.