Elsevier

Biomaterials

Volume 30, Issue 22, August 2009, Pages 3637-3644
Biomaterials

Cell attachment functionality of bioactive conducting polymers for neural interfaces

https://doi.org/10.1016/j.biomaterials.2009.03.043Get rights and content

Abstract

Bioactive coatings for neural electrodes that are tailored for cell interactions have the potential to produce superior implants with improved charge transfer capabilities. In this study synthetically produced anionically modified laminin peptides DEDEDYFQRYLI and DCDPGYIGSR were used to dope poly(3,4-ethylenedioxythiophene) (PEDOT) electrodeposited on platinum (Pt) electrodes. Performance of peptide doped films was compared to conventional polymer PEDOT/paratoluene sulfonate (pTS) films using SEM, XPS, cyclic voltammetry, impedance spectroscopy, mechanical hardness and adherence. Bioactivity of incorporated peptides and their affect on cell growth was assessed using a PC12 neurite outgrowth assay. It was demonstrated that large peptide dopants produced softer PEDOT films with a minimal decrease in electrochemical stability, compared to the conventional dopant, pTS. Cell studies revealed that the YFQRYLI ligand retained neurite outgrowth bioactivity when DEDEDYFQRYLI was used as a dopant, but the effect was strongly dependant on initial cell attachment. Alternate peptide dopant, DCDPGYIGSR was found to impart superior cell attachment properties when compared to DEDEDYFQRYLI, but attachment on both peptide doped polymers could be enhanced by coating with whole native laminin.

Introduction

Modification of conventional platinum (Pt), gold or iridium oxide electrodes with conducting polymer coatings has the potential to significantly improve the long-term performance of neural implants including the cochlear implant, vision prosthesis, neural regeneration devices and neural recording electrodes [1], [2]. Conventional electrode materials are typically fabricated with smooth surfaces that are not conducive to tissue integration. As a result the interface between a metal electrode and neural tissue is associated with a significant fluid gap through which the electrical signal must be transduced. This distance is critical in determining the current amplitude required to activate the neural tissue and the quality of the perceived signal [3], [4].

Conducting polymers such as polypyrrole (PPy) and polythiophene derivative poly(3,4-ethylene dioxythiophene) (PEDOT) have been used by several research groups to enhance the properties of neural interfaces [5], [6], [7], [8], [9]. Conducting polymer coatings have been shown to improve the charge transfer characteristics of conventional metal electrodes and biological assays have shown that cells preferentially adhere to coated electrodes [5], [10], [11]. The biological interaction has the potential to eliminate the fluid gap through intimate contact between the tissue and electrode. It is hypothesised that controlled interaction between the conducting polymer and surrounding tissue can be achieved through the incorporation of biological molecules tailored to produce a response from specific cell types [2].

Extracellular matrix molecules are known to support cell attachment and growth when incorporated into conducting polymers or used as a coating. Molecules that have been implicated in these roles for cell regeneration include laminin, chondroitin sulfates, other proteoglycans and hyaluronic acid (HA) [12], [13], [14]. Laminin, a multidomain basement membrane glycoprotein is known to provide a permissive substrate that binds to cell surface receptors and also can function to stimulate neurite extension [15], [16]. However, the use of the full multidomain protein or even a single domain bears some disadvantages including the need for isolation and purification, the risk of degradation via immune attack and proteolysis [17]. Only specific sections of the laminin molecule contain the required receptors for cell adherence and studies have shown that partial functions of the large laminin molecule can be imitated by smaller fragmental components with specific functional binding [18]. Investigations by Huber et al. have found that synthetically prepared peptides have a greater stability than native laminin [19].

Peptides of laminin can be synthetically manufactured and tailored for incorporation into conducting polymers. The peptide sequence is determined from the desired cell response. Laminin peptides have been reported as having specific cell response ligands with several domains having been identified for cell attachment or neural cell growth and development [15], [20], [21], [22], [23], [24]. The addition of various amino acids to the end of a linear peptide chain can be used to control the overall ionic behaviour of the molecule. This ability to produce tailored laminin peptides as anions makes them ideal candidates for doping common conducting polymers.

While a number of cell attachment proteins have been incorporated into conducting polymers or onto the polymer surface, the effect of these molecules on polymer properties is not defined. In studies by Cui et al. synthetically manufactured DCDPGYIGSR was incorporated into PEDOT derivative PEDOT-MeOH films as a dopant, and showed good cell attachment properties, but was not characterised for electrochemical stability, mechanical hardness and adhesion to the electrode surface [13]. In order to produce a long-term durable implant, the impact of large biomolecules on polymer structures needs to be explored [2].

This study investigates the use of two different laminin peptide sequences in doping PEDOT. The resulting polymers are characterised across a range of properties integral to their long-term performance in a neuroprosthetic device. Two peptide sequences derived from laminin were synthetically manufactured with the addition of amino acids to produce anionic peptides. DCDPGYIGSR was chosen due to positive results reported by Cui et al. [10]. DEDEDYFQRYLI, a peptide which has not previously been incorporated into conducting polymers, was also assessed. This peptide contains the active sequence YFQRYLI. The YFQRYLI ligand was identified by Tashiro et al. and reported to mediate cell attachment and promote neurite outgrowth in both PC12 cells and cerebellar microexplant cultures [22]. In this study it was hypothesised that the addition of a DEDED anionic tail to YFQRYLI would produce a molecule capable of doping PEDOT, while providing increased cell adhesion and supporting neurite outgrowth.

The aim of this study was to produce conducting polymer coatings of PEDOT doped with synthetic anionic laminin peptides, DCDPGYIGSR and DEDEDYFQRYLI on model Pt electrodes. The effect of a large biomolecule dopant on conducting polymer physico-chemical properties was established with comparison to conventionally doped PEDOT/paratoluene sulfonate (pTS). The effect of peptide doped polymers on mammalian cell interactions and the cell response to peptides containing specific bioactive ligands was assessed using the neural-like PC12 cell line.

Section snippets

Electropolymerisation

For each electrodeposition 0.1 m EDOT (Cat # 483028 Sigma–Aldrich) doped with 5 mg/mL of synthetic peptide was made up in a 1 part acetonitrile: 1 part deionised (DI) water solution. DCDPGYIGSR (10-mer, 1 kDa) and DEDEDYFQRYLI (12-mer, 1.6 kDa) custom peptides were produced by Invitrogen. Control films of 0.1 m EDOT doped with 0.05 m pTS (Cat # 152536 Sigma–Aldrich) were produced under identical conditions. Polymers were electrodeposited onto Pt electrodes using an in-house manufactured galvanostat

Results

All polymers fabricated on the Pt foil were dark blue, confluent and appeared uniform when viewed macroscopically. During deposition it was noted that the electrolyte solution from which both the peptide doped films were evolved turned the same blue colour as the polymerised film, indicating that the polymer did not only form on the working electrode, but was dispersed throughout the electrolyte.

SEMs representative of the overall polymer surface morphology are shown in Fig. 1. The most notable

Discussion

PEDOT was doped with laminin peptides DEDEDYFQRYLI and DCDPGYIGSR through electropolymerisation using conventional deposition parameters for bioactive conducting polymers [10], [27], [28]. The resulting film was less uniform in microscopic appearance than the control PEDOT/pTS film, and physico-chemical properties were affected by the larger dopant. It was found that the peptide dopants produced bioactive films with properties relevant to the specific ligands, but they were not able to promote

Conclusions

The incorporation of laminin peptides into PEDOT has revealed that these potential dopants significantly impact on polymer physico-chemical properties. The use of large synthetic peptides as anionic dopants produced a softer interface with improved impedance characteristics, especially in the low frequency, biologically significant region. However, reduced polymer electrochemical stability and lower adherence of the films to the Pt electrode was observed when compared to the conventionally

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