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. 2010 Nov;6(11):4396-404.
doi: 10.1016/j.actbio.2010.06.011. Epub 2010 Jun 15.

Pyrrole-hyaluronic acid conjugates for decreasing cell binding to metals and conducting polymers

Jae Young Lee  1 , Christine E Schmidt
Affiliations

Pyrrole-hyaluronic acid conjugates for decreasing cell binding to metals and conducting polymers

Jae Young Lee et al. Acta Biomater. 2010 Nov.

Abstract

Surface modification of electrically conductive biomaterials has been studied to improve biocompatibility for a number of applications, such as implantable sensors and microelectrode arrays. In this study we electrochemically coated electrodes with biocompatible and non-cell adhesive hyaluronic acid (HA) to reduce cellular adhesion for potential use in neural prostheses. To this end, pyrrole-conjugated hyaluronic acid (PyHA) was synthesized and employed to electrochemically coat platinum, indium-tin oxide and polystyrene sulfonate-doped polypyrrole electrodes. This PyHA conjugate consisted of (1) a pyrrole moiety that allowed the compound to be electrochemically polymerized onto a conductive substrate and (2) non-adhesive HA to minimize cell adhesion and to potentially decrease inflammatory tissue responses. Our characterization results showed the presence of a hydrophilic p(PyHA) layer on the modified electrode, and impedance measurements revealed an impedance that was statistically the same as the unmodified electrode. We found that the p(PyHA)-coated electrodes minimized adhesion and migration of fibroblasts and astrocytes for a minimum of up to 3 months. Also, the coating was stable in physiological solution for 3 months and was stable against enzymatic degradation by hyaluronidase. These studies suggest that this p(PyHA) coating has the potential to be used to mask conducting electrodes from adverse glial responses that occur upon implantation. In addition, electrochemical coating with PyHA could potentially be extended for the surface modification of other metallic and conducting substances, such as stents and biosensors.

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Figures

Figure 1
Figure 1
(A) The chemical structure of the PyHA conjugate synthesis. PyHA conjugate was synthesized by coupling 1-aminopropyl pyrrole to the carboxylic groups of HA. (B) Schematic of the PyHA conjugate illustrating the degree of substitution of the HA with the pyrrole. 1H NMR analysis indicated that approximately 5-15% of carboxylic groups were modified with pyrrole.
Figure 2
Figure 2
Voltammogram of the potentiodynamic synthesis with PyHA (red lines) on ITO in 0.5 wt% aqueous polymer solution. Twenty cycles from 0 V to 1.0 V, versus SCE, were applied with a scan rate of 0.1 V s-1. The same electrochemical procedure was repeated using unmodified HA solution (black lines). Electrochemical oxidation was observed above 0.8 V with PyHA, but not with unmodified HA.
Figure 3
Figure 3
High resolution (A) C1s and (B) N1s XPS spectra of bare ITO and p(PyHA)-coated ITO. After p(PyHA) coating, distinct changes in spectra were observed, suggesting the presence of the p(PyHA) on surfaces.
Figure 4
Figure 4
(A) AFM images of bare Pt and HA-coated Pt substrates. The p(PyHA) coating of Pt decreased surface RMS roughness, but formed nano-clusters on the surfaces. (B) The border between bare Pt and the HA-coated area was scanned, indicating a thickness (Δx) of 20-40 nm thickness for dry films.
Figure 5
Figure 5
(A) Magnitudes and (B) phase angles of impedance for unmodified Pt and p(PyHA)-coated Pt. Impedance spectra were collected in a range of 1-105 Hz, applying an AC sinusoidal signal with 10 mV, vs SCE, in PBS solution. Averages and standard deviations were obtained from three samples for each condition.
Figure 6
Figure 6
In vitro cell culture on bare ITO and p(PyHA)-coated ITO. (A) Normal human dermal fibroblast cells (nHDF) were cultured for 3 days and stained for F-actin (green) and nuclei (blue). The nHDF cells did not adhere to the p(PyHA)-coated substrates. (B) Cortical astrocytes were also cultured for 3 days on ITO, p(PyHA)-coated ITO, and HAase-treated p(PyHA)-coated ITO, followed by immunostaining for GFAP (green, astrocyte marker), nuclei (blue), and HA (red). Astrocytes did not grow on p(PyHA)-coated surfaces; however, removal of surface p(PyHA) with HAase permitted adhesion and growth comparable to bare electrodes. Scale bars are 50 μm.
Figure 7
Figure 7
Phase contrast images of astrocytes cultured on Pt and PPyPSS substrates either unmodified or coated with p(PyHA). Astrocytes did not grow on surfaces with the p(PyHA)-coating, whereas unmodified substrates permitted cell adhesion and growth. Scale bars are 50 μm.
Figure 8
Figure 8
Functional stability of the p(PyHA) coating. (A) The p(PyHA)-coated ITO was incubated in PBS at 37°C for 3 months, followed by astrocyte culture on the incubated substrates. The dashed line indicates the border between the HA-coated area and the unmodified area. The p(PyHA) coating (red) was stable and resisted adhesion of astrocytes (green for GFAP and blue for nuclei). (B) Long-term astrocyte culture on HA-coated Pt for 1 month. Cells did not attach or migrate to HA-coating during cell culture. (C) Long-term astrocyte culture on HA-patterned ITO for 3 months; images were taken at same location for all time points, showing no adhesion on and migration to the HA-patterned area for 3 months. Scale bars are 50 μm.
Figure 9
Figure 9
Enzymatic stability tests of the p(PyHA)-coated ITO. (A) Water contact angles were measured for the HAase-treated substrates (n=3). (B) Immunofluorescence intensities were measured from the HAase-treated substrates (n=3). The HAase-treated substrates were stained with bHABP and streptavidin-PE. All substrates were treated and imaged at the same conditions. (C) Astrocytes were cultured on the HAase-treated p(PyHA) substrates for 3 days and numbers of nuclei were counted (n=5). Averages ± standard deviations are plotted.
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