Conducting polymers, dual neurotrophins and pulsed electrical stimulation — Dramatic effects on neurite outgrowth

Abstract

In this study the synergistic effect of delivering two neurotrophins simultaneously to encourage neuron survival and neurite elongation was explored. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) were incorporated into polypyrrole (PPy) during electrosynthesis and the amounts incorporated and released were determined using iodine-125 (¹²⁵I) radio-labelled neurotrophins. Neurite outgrowth from cochlear neural explants grown on the conducting polymer was equivalent to that on tissue culture plastic but significantly improved with the incorporation of NT-3 and BDNF. Neurite outgrowth from explants grown on polymers containing both NT-3 and BDNF showed significant improvement over PPy doped only with NT-3, due to the synergistic effect of both neurotrophins. Neurite outgrowth was significantly improved when the polymer containing both neurotrophins was electrically stimulated. It is envisaged that when applied to the cochlear implant, these conducting and novel polymer films will provide a biocompatible substrate for storage and release of neurotrophins to help protect auditory neurons from degradation after sensorineural hearing loss and encourage neurite outgrowth towards the electrodes.

Introduction

The use of conducting polymers to promote neurite outgrowth has attracted considerable attention over the past 15 years. A number of studies [1], [2], [3], [4] have shown that it is possible to facilitate neurite outgrowth on conducting polymer platforms. Shastri et al. [5] showed that neurite extension from PC-12 cells was facilitated on polypyrrole (PPy) surfaces and that the application of an electrical stimulus to the PPy film significantly increased the neurite formation. Others [4] have also reported a significant increase in neurite length when electrical stimulation was applied to a PPy platform supporting nerve cell growth. It was subsequently shown that electrical stimulation of PPy increases the absorption of fibronectin (an extracellular matrix glycoprotein) from the cell culturing media onto the conducting polymer surface [3]. Since nerve cells are known to interact with fibronectin [6], this is undoubtedly an important step in cell attachment to the conducting polymer.

Hyaluronic acid is also known to provide anchoring sites for nerve cells [7]. For this reason, either hyaluronic acid (HA) (1) or sulfonated HA (2) have been covalently attached to a PPy substrate to improve adhesion of PC-12 cells [8]. Both surface modifications also increased blood compatibility compared with the pristine PPy surface. Similarly, modification of PPy with poly(vinyl alcohol) improved attachment of PC-12 cells and enhanced biocompatibility as evidenced by decreased adsorption of fibrinogen on the modified conducting polymer surface compared to PPy itself [9].

However, direct covalent attachment of moieties to a conducting polymer backbone usually has an adverse effect on the electrical properties of the polymer. Incorporation of the biomolecule of interest as the dopant anion is therefore an attractive alternative. Studies have shown that biomolecules retained in the conducting polymer matrix have a dramatic influence on the ability to effectively interact with neurons. PPy doped with a modified silk-like protein F (Pronetin F) that contains fibronectin fragments was found to interact more effectively with rat glial cells than unmodified PPy. In the same study, incorporating the nona-peptide (CDPGYIGSR) was shown to improve interactions with human neuroblastoma cells [5]. Incorporation of the bioactive molecule as the dopant has the added benefit that combinations of molecules are readily incorporated during polymer synthesis. For example, Stauffer and Cui [10] have shown that the incorporation of two laminin fragments (CDPGYIGSR and RNIAEIIKDI) supported the growth of high neuronal density and decreased astrocyte adhesion (compared to a simple gold electrode) in culture.

The incorporation of nerve growth factor (NGF) molecule into PPy has also been shown to be beneficial to nerve survival and growth [1]. In the case of NGF, the molecule is positively charged at the pH used for polymerisation, therefore incorporation is facilitated by the use of an anionic polyelectrolyte dopant that acts as a molecular carrier facilitating protein entrapment. While much of the recent research into the controlled release of proteins utilise hydrogel [11], [12] or polymer entrapment [13], [14] the use of PPy as a release matrix for proteins can offer advantages in terms of electrically-controllable rate of delivery. Additionally, the use of electrical stimulation to delivery is expected to have an additional positive effect on target neural cells for neural engineering applications, particularly in the cochlear implant.

Incorporation/entrapment of the growth factor neurotrophin-3 (NT-3) into PPy during polymerisation has also been achieved [15]. It was established that the electrically stimulated release of NT-3 at functional rates could be effected using clinically acceptable stimulation protocols (i_d = ± 1.0 mA, pulse width 100 μs, frequency 250 Hz) [16], [17] The use of this neurotrophin loaded platform to sustain the growth of auditory neurons in cultured cochlear tissue and to promote neurite outgrowth from these structures upon electrical stimulation was subsequently demonstrated [16].

The use of two neurotrophins, namely brain-derived neurotrophic factor (BDNF) and NT-3, for rescue of auditory neurons in vivo has been investigated using osmotic pumps or bolus delivery to introduce the therapeutic proteins into the cochlea. NT-3 and BDNF are ligands for Trk receptors expressed on cochlear neuron surfaces, binding to the cell receptor to trigger cellular responses by activating the cell machinery. Delivery of BDNF alone to the cochlea of deafened guinea pigs led to survival of 80% of auditory neurons, compared to 30% survival for untreated cochleae [18], [19], [20], [21]. Combining NT-3 and BDNF delivery to the cochlea has been found to enhance auditory neurons survival even more effectively than delivery of either neurotrophin alone [22], [23], [24], presumably due to their separate action on two different cell receptors to trigger cell signalling cascades promoting the survival and differentiation of the nerve cells. Additionally, it has previously been reported that there is a synergistic effect on survival of auditory nerves with electrical stimulation and addition of neurotrophins, wherein providing both signals to cells led to a response greater than the expected additive effects of either treatment alone [25].

Here we report the incorporation and localised release of dual neurotrophins BDNF and NT-3 from the conducting polymer PPy and the dramatic synergistic effect on neurite outgrowth from cochlear neural explants. Based on previous research, it was expected that if BDNF and NT-3 could be simultaneously released in an electrically-controlled manner from PPy, significant benefits should be obtained over release of single neurotrophins as previously reported.