116 (2006) 285–294www.elsevier.com/locate/jconrel

Journal of Controlled Release

Optimising the incorporation and release of a neurotrophic factor usingconducting polypyrrole

Brianna C. Thompson a,b, Simon E. Moulton a,b, Jie Ding a,b, Rachael Richardson c,Adrian Cameron a,c, Stephen O'Leary c, Gordon G. Wallace a,b,⁎, Graeme M. Clark a,c

a ARC Centre of Excellence for Electromaterials Science, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australiab Intelligent Polymer Research Institute, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia

c Bionic Ear Institute, University of Melbourne, 384–388 Albert St, East Melbourne, VIC 3002, Australia

Received 5 April 2006; accepted 13 September 2006Available online 29 September 2006

Abstract

In this study, a neurotrophin delivery system based on an inherently conducting polymer (ICP) has been developed. Direct incorporation ofneurotrophin-3 (NT-3) was investigated and controlled release was tested under various electrochemical conditions. The loading capacity andamount of NT-3 released from the polymer was determined using 125I-labelled NT-3. Electrochemical stimulation of polypyrrole by pulsedvoltage, pulsed current or cyclic voltammetry promoted the release of NT-3 at a greater rate than natural diffusion of NT-3. NT-3 was releasedfrom polypyrrole as an initial burst in the first 24 h followed by prolonged release over a subsequent 6 days of sampling. The amount of NT-3incorporated into the polymer could be controlled by varying the polymerisation time, with longer growth periods incorporating more NT-3. TheNT-3 release results indicated that the polymers grown for longer released a lower percentage of the incorporated NT-3 compared to the polymersgrown for shorter times. Polymer-based neurotrophin delivery systems have the potential to be incorporated into future treatments for nerveinjuries to prevent nerve degradation and promote nerve protection.© 2006 Elsevier B.V. All rights reserved.

Keywords: Polypyrrole; Neurotrophin-3; Electrochemical; Neurons; Controlled release

1. Introduction

Inherently conducting polymers (ICPs) have been widelystudied for use as biomaterials for tissue repair and cell growthdue to the inherent ability of these polymers to locally deliver anelectrical stimulus to the cells [1–4]. Polypyrrole in particularhas been shown to be biocompatible and has been proposed forseveral in vivo applications [1,5–9]. The use of polypyrrole as amaterial for the controlled release of clinically relevant amountsof a neurotrophin is described here. The purpose of this researchis to develop new materials to assist in preventing nervedegradation and promoting nerve protection, particularly for usewith the cochlear implant. This body of work constitutes the

⁎ Corresponding author. ARC Centre of Excellence for ElectromaterialsScience, University of Wollongong, Northfields Avenue, Wollongong, NSW2522, Australia. Tel.: +61 2 4221 3127; fax: +61 2 42213114.

E-mail address: gwallace@uow.edu.au (G.G. Wallace).

0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2006.09.004

first phase in the development of an implantable device capableof delivering a neurotrophic factor specifically at the site ofnerve damage within the cochlear.

The synthesis of polypyrrole can be achieved using eitherchemical or electrochemical oxidation, with a counter ion(molecular dopant) being incorporated into the polymer duringgrowth. A wide range of molecular dopants such as DNA,antibodies, enzymes, growth factors and even whole living cells[10] can be used. The dopant molecule (A−: Scheme 1A)determines the mechanical, electrical and chemical properties ofthe resulting polymer. Redox cycling of ICPs (Scheme 1B) canresult in the incorporation and release of various molecules,both positively and negatively charged [11]. It has also beenshown that from careful choice of the dopant anion it is possibleto induce cation incorporation and release upon reduction andoxidation of the conducting polymer [12]. This may allowconstruction of a biocompatible controlled release device todeliver various drugs or biomolecules, such as neurotrophins.

Scheme 1. (A) Synthesis of ICP showing the incorporation of the dopant A−. (B)Release of the dopant A− during redox cycling of the ICP.

286 B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

A variety of novel materials and strategies have been de-veloped that provide support for neuronal growth [13] or as drug[13,14], hormone [15] and gene [16] delivery systems. Benoit etal. [17] developed biodegradable glial cell line-derived neuro-trophic factor (GDNF) loaded PLGAmicrospheres, which couldbe implanted by stereotaxy in the brain and could offer analternative strategy in the treatment of Parkinson's disease.These microspheres showed promising release profiles over a56 days period with the bioactivity of the GDNF confirmed. Acommon problem associated with long-term implantable neuraldevices is the loss of connectivity due to post implantationinflammatory reaction. To overcome the issue Bellamkonda andZhong [18] developed a novel nitrocellulose-based implantcoatings for the sustained delivery of the anti-inflammatoryneuropeptide α-melanocyte stimulating hormone (α-MSH). Theα-MSH was incorporated into micron-scale nitrocellulosecoatings and slow, sustained release over 21 days was attainedin vitro at which point the α-MSH bioactivity was confirmed.While this coating was effective at releasing the anti-inflammatory hormone the ability of the coating to inhibit theinflammatory reaction was not tested. The issue of addressing theinflammatory response of implanted devices has been addressedmore recently [19]. Cui et al. [19] used polypyrrole to incorporateand release the anti-inflammatory drug dexamethasone in orderto inhibit astro-glial sheath formation. This sheath formation isalso commonly attributed to the failure of implanted micro-machined neural electrode arrays. The continued improvementof the interfacial contact between electrode sites and biologicaltissue has been a key area of research in the development ofneural prosthetic devices [20]. Cui et al. [20] showed that byincorporating appropriate biomolecules into conducting poly-pyrrole grown on multichannel neural probes it was possible toimprove the adhesion of glial and human neuroblastoma cells.The presence of the polypyrrole resulted in a decrease in surfaceimpedance for the multichannel neural probes [20–22].

Using either in vitro or in vivo experiments the ability ofneurotrophins to prevent the degeneration of auditory neurons[23] has been demonstrated. Neurotrophins have been associ-ated with roles in both developing and mature neuron cellpopulations; promoting differentiation, preventing neuron celldeath, and maintaining nerve cell function [24–27]. In addition,two key neurotrophins – neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) – have been shown toinduce neurite outgrowth and enhance auditory neuron survival

in vitro [28]. NT-3 is a protein which is secreted into theextracellular environment by various neural cells and nervetarget tissues, where it interacts with the extracellular trk familyof tyrosine kinase receptors [29]. The neurotrophin–receptorcomplex can then undergo receptor-mediated endocytosis intovesicles from the nerve terminal, where it is transported to thecell body [30]. Once in the cell nucleus, neurotrophin-3 can elicitbiological responses, such as promoting neural survival andneurite outgrowth. The minimum amount of NT-3 required topromote growth and survival of various nerve cells is not welldocumented.

The ability to promote neural growth, or at least enhancenerve cell survival, has important implications in nerveregeneration as well as for interfacing with bionic structures.Controlled release of neurotrophins has been proposed as a vitalcomponent in nerve regeneration, where the neurotrophins actwith high potency in low concentrations to prevent nerve celldeath, encourage nerve growth, and even control direction ofnerve fibre growth [31]. It has also been found that smallelectrical pulses, in combination with a supply of growth factors,improve the survival and growth of nervous tissue [32,33].Inherently conducting polymers such as polypyrrole canpotentially provide electrical stimulation and release growthfactors simultaneously, making them a most useful platform forpreventing nerve degradation and promoting nerve protection[6,9,34,35]. An immediate application of the neurotrophin-releasing conducting polymer lies in the cochlear implant, whereloss of nerve cells after hair cell loss is a major problem, andrelease of neurotrophin may help to prevent nerve recessionaway from the electrode. The work described here has beenperformed specifically for this application.

In this study, we incorporated radiolabelled (125I) neuro-trophin-3 (125I-NT-3) into polypyrrole and induced controlledrelease of the neurotrophin under specific electrochemicalconditions. The effect of polymer synthesis conditions and thesubsequent electrical stimulation used on the release propertieshas been investigated.

2. Experimental

2.1. Reagents

Pyrrole (Py) was obtained from Merck and distilled prior touse. para-Toluene sulphonic acid, sodium salt (pTS) wasobtained from Aldrich and was of analytical reagent (AR)grade. NT-3 was obtained from Chemicon International.Iodination of NT-3 was performed by ProSearch InternationalAustralia. NT-3 has a molecular weight of approximately13.6 kDa and a pI=9.2, making it positively charged at the pHused in the work (pH=7.2–7.4). The hydrodynamic radius ofNT-3 is approximately 66 Å. Deionised Milli-Q water (18 MΩcm−1) was used to prepare all solutions. Gold coated Mylar(18 Ω/square) was purchased from CPFilms Inc (USA).

The controlled release of the incorporated 125I-NT-3, hereafter only referred to as NT-3, was performed in a 0.9% NaClsolution. The pH of the solution was adjusted to 7.2–7.4 withKOH. The solution was 0.2 μm filtered, and stored at 4 °C.

287B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

2.2. Instrumentation

Synthesis of polymers was performed galvanostatically in atwo-electrode (working and auxiliary electrode) cell using a 363model potentiostat/galvanostat (EG and G Princeton AppliedResearch), controlled by EDAQ software via aMacLab II. In thecontrolled release of NT-3, electrochemical parameters werecontrolled and recorded in a three electrode (working, auxiliaryand reference electrode) cell using EDAQ software via aMacLab II, and a 363 model potentiostat/galvanostat. Themorphology of the polymers was characterised using a LeicaStereoscan 440 SEM model scanning electron microscope(SEM). All radiation measurements were performed using aCobra 5000 γ-counter with a 125I detection efficiency ofapproximately 75%. Thickness data was obtained using aDektak 6MBench-Top Surface Profiler manufactured byVeeco.

2.3. Growth of polypyrrole/pTS (PPy/pTS) with NT-3incorporated

A two-electrode electrochemical cell was set up in a1 cm×1 cm plastic cuvette, with stainless steel mesh as anauxiliary electrode, and gold-Mylar (masked to 1 cm2 area) asthe working electrode. Polymer containing NT-3 (PPy/pTS/NT-3)was galvanostatically grown at 2 mA (2 mA/cm2) using atwo-layer approach. The development of this approach isdescribed below (Section 3.3). The first layer was grown for90 s (estimated thickness=0.4 μm) from 0.2 M pyrrole and0.05 M pTS at 2 mA/cm2, controlled by a 363 modelgalvanostat/potentiostat (EG and G Princeton Applied Re-search). After this, the PPy/pTS polymer-covered workingelectrode was transferred to an identical electrochemical cellconfiguration containing 0.2 M pyrrole and 0.05 M pTS and anappropriate concentration of NT-3. Due to the 125I radiolabellingprocess the exact concentration of NT-3 varied from batch tobatch. It was therefore necessary to average out the NT-3concentration received in the various radiolabelled batches. Theaverage NT-3 concentration for all of the received radiolabelledbatches was calculated to be 1.35 (±0.08) ppm. Throughout thispaper the NT-3 concentration will be reported as 1.35 ppm. Thesecond layer was then grown for either 10 or 60 min. Uponcompletion of the polymerisation the polymer was washedthoroughly under a stream of Milli-Q water.

2.4. Release of NT-3 from polymers

Four different release protocols were investigated — pulsedpotential, pulsed current (both ±0.5 mA and ±20 mA) at 5 Hzand scanning the voltage between two pre-defined voltage

Table 1Conditions used for each method of NT-3 release into 0.9% NaCl solution

Release method Stimulus applied Frequency or scan rate

Cyclic voltammetry −0.8 /+1.0 V 50 mV/sPulsed potential −0.6 /+0.6 V 5 HzPulsed current (±0.5 mA) −0.5 /+0.5 mA 5 HzPulsed current (±20 mA) −20 /+20 mA 5 Hz

limits at a set scan rate (cyclic voltammetry). Each releaseprotocol was continuously applied to the PPy/pTS/NT-3samples throughout the duration of the release experiments.Table 1 shows the details of each release method used. Each wasperformed in an identical three electrode cell, with the workingelectrode used being the PPy/pTS/NT-3 coated gold-Mylarstrip, the counter electrode being stainless steel mesh, and thereference electrode being Ag/AgCl (3.0 M NaCl). Theelectrolyte used for the release cell was 0.9% NaCl. The releaseconditions were controlled and monitored by EDAQ softwareutilizing either Chart v5.1.2 or EChem v2.0.2 via a MacLab IIand a Princeton Applied Research PAR-363 galvanostat/potentiostat.

Each release experiment was performed in triplicate, with thesamples analysed using a Cobra 5000 Gamma Counter. After12 min under the release conditions, the release media wascompletely removed from the cell, and placed into a scintillationvial. Fresh 0.9% NaCl media was replaced and the release wascontinued for a further 48 min. After this, the media wascollected again, as well as the working electrode (PPy/pTS/NT-3),and both were placed separately into scintillation vials. The12 minute, 48 minute, and working electrode (PPy/pTS/NT-3)samples were gamma counted for 5 min, from which an un-corrected counts per minute (CPM) value was obtained. Thisvalue was corrected for the decay rate of 125I, and the mass ofNT-3 incorporated and released from the polymer was cal-culated using the specific activity of the labelled NT-3, and thefollowing equations;

TotalNT�3 incorporated in polymer¼ amount NT�3 in polymerþ amount NT�3 released in 12min sampleþ amount NT�3 released in 48min sample

ð1Þ

12min release ¼ amount NT�3 released in 12min ð2Þ

60min releaseð0total release0Þ¼ amount NT�3 released in12min sampleþ amount NT�3 released in 48min sample

ð3Þ

3. Results and discussion

3.1. Synthesis of PPy/pTS/NT-3

Initially cyclic voltammetry (CV) was used to study theelectropolymerisation process (data not shown). Using thepolymerisation solution containing just pyrrole and pTS(described in the Experimental section) it was found thatpyrrole was oxidised at approximately + 0.8 V (vs. Ag/AgCl).The increase in current magnitude observed in subsequentpotential scans indicates the deposition of a conductingelectroactive polymer. Cyclic voltammograms (CVs) recordedin a pyrrole/pTS monomer solution containing NT-3 were

Fig. 2. Film thickness for polymers grown under galvanostatic conditions forvarious times from a monomer solution containing 0.2 M pyrrole/0.05 M para-Toluene sulfonate and 1.35 ppm NT-3.

288 B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

identical to the CVs recorded with no NT-3 in the monomersolution and resulted in the deposition of polymer films,indicating that the NT-3 containing polymers (see later) weresimilar with respect to electronic and electrochemical proper-ties. However, the polymer film deposited in the presence ofNT-3 was brittle to touch and delaminated from the underlyinggold coated Mylar upon handling. Previous studies had shownthat galvanostatic control is usually the preferred electrochem-ical method of growth with respect to obtaining polypyrrolefilms that exhibit good mechanical strength as well as superioradhesion to the underlying substrate [36]. In other studies theoptimal current density was 2 mA/cm2, resulting in theproduction of a mechanically robust PPy/pTS polymer [37].Previously published work [38] and our preliminary workindicated that a ratio of 0.2 M pyrrole to 0.05 M pTS produced apolymer with superior mechanical properties and adhesion tothe underlying substrate.

In the present study the use of galvanostatic growthconditions with a current density of 2 mA/cm2 resulted in thedeposition of a conducting electroactive layer. During poly-merisation a constant potential of + 0.85 V (vs. Ag/AgCl) wasobtained, confirming the deposited film to be in the conductingand electroactive form. This conductivity and electroactivitywas further confirmed from cyclic voltammograms obtainedusing the film as the working electrode after growth (Fig. 1).The responses (A/A′) observed in the CV can be attributed tothe oxidation and reduction respectively of the polypyrroleaccording to the process shown in Scheme 1B. The filmsdeposited were uniform and adherent.

3.2. Optimising polymer growth conditions

The current density of 2 mA/cm2 was chosen as this has beenshown to produce mechanically robust polypyrrole films [38].The polymers were grown in a two-electrode cell. It was foundthat the use of current densities greater than 2 mA/cm2 resultedin the formation of an over-oxidised polypyrrole film withreduced conductivity [39]. It was also observed that for sixseparate samples, three each being prepared at 1 mA/cm2 and2 mA/cm2 that latter current density produced slightly higher

Fig. 1. Cyclic voltammogram of PPy/pTS/NT-3 polymer recorded in 0.2 M PBS(pH 7.4) between −0.8 Vand +0.8 Vat a scan rate of 100 mV/s. PPy/pTS/NT-3grown at 2 mA/cm2 for 60 min in a monomer solution containing 0.2 M pyrrole,0.05 M pTS and 1.35 ppm NT-3. Arrows indicate the direction of potential scan.

NT-3 incorporated amounts (22 ng/cm2 at 2 mA/cm2 vs. 20 ng/cm2 at 1 mA/cm2). Consequently, a current density of 2 mA/cm2 was employed throughout this study, unless otherwisestated. However, under these conditions it was not possible toobtain a continuous polymer film with every synthesis;therefore a two-layer polymerisation approach was developedto overcome this. The patchy growth of the polymer on baregold suggests that the inclusion of NT-3 into the monomersolution influences the polymerisation process. When a goldMylar sample was placed into a solution of 1.35 ppm NT-3 for30 s followed by thorough rinsing with Milli-Q water a totalamount of 1.8 ng/cm2 of NT-3 was measured on the gold Mylarsurface. This result indicates that the NT-3 adsorbs onto the goldMylar surface causing biofouling, which may be the cause ofthe non-uniform polymer film formation. Adsorption of proteinonto gold electrodes has been reported previously [40].

3.3. Two-layer approach to polymer growth

In order to obtain a continuous PPy/pTS/NT-3 polymer ongold-coated Mylar with higher loading of NT-3, a two-layerapproach was developed. The first layer consisted of a polymergrown from 0.2 M pyrrole and 0.05 M pTS at a current densityof 2 mA/cm2 for 90 s. This layer was deposited as a thincontinuous film. The second layer was deposited from amonomer solution of 0.2 M pyrrole, 0.05 M pTS and 1.35 ppmNT-3 grown at 2 mA/cm2 for various times. The presence of thispre-layer did not affect the potential drawn during the polymergrowth. The two-layer approach may improve polymer growthby shielding the gold surface from the biomolecule, or may havean effect due to the increased surface roughness, and thus highersurface area of the electrode area. The thickness of the PPy/pTS/NT-3 films grown under galvanostatic methods were measuredusing a Dektak 6 M Stylus Profiler with the results shown inFig. 2. The polymer film thickness increased as the polymer-isation time increased.

The amount of NT-3 incorporated using the two-layerapproach was found to be slightly greater (25 ng/cm2 comparedto 20 ng/cm2 for 10 min growth) than when deposited as acontinuous film directly on the gold coated Mylar substrate. A

Fig. 3. SEM images of PPy/pTS/NT-3 films (A) before and (B) after 60 minelectrical stimulation in 0.9% NaCl solution. The polymers were grown using a2-layer approach for 60 min at 2 mA/cm2 (26 μm thickness) from a monomersolution containing 0.2 M pyrrole, 0.05 M pTS and 1.35 ppm NT-3. Theelectrical stimulation protocol used was ±0.5 mA/cm2 at a frequency of 5 Hz.

Fig. 4. The effect of polymerisation time on the amount (ng/cm2) of NT-3incorporated into the PPy/pTS polymer. Polymerisation was performed using acurrent density of 2 mA/cm2 with monomer/dopant concentrations of 0.2 Mpyrrole/0.05 M pTS/1.35 ppm. The equation shown is of the linear regressiontrendline fit. The error bars represent the standard error.

289B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

two-layer approach was also employed by Collier et al. [6] toproduce a polypyrrole composite material suitable for use intissue engineering applications. Under all experimental condi-tions the second layer, containing the NT-3, deposited as acontinuous layer, confirmed by SEM (Fig. 3). SEMs obtainedfor the PPy/pTS/NT-3 film showed the cauliflower morphologyusually observed for this polymer. No differences wereobserved in SEMs obtained for the polymer containing noNT-3 (not shown).

The effect of polymerisation time on the amount of NT-3incorporated into the polymer was also investigated. Theamount of NT-3 incorporated into the polymer increasedlinearly with increased polymerisation time (Fig. 4). Themaximum growth period of 60 min produced adherent uniformfilms with polymer NT-3 loading that was deemed high enoughfor use in a controlled release device [31]. The optimalmonomer solution conditions required to obtain sufficient NT-3 loading was determined to be 0.2 M pyrrole, 0.05 M pTS and1.35 ppm NT-3. All subsequent PPy/pTS/NT-3 polymerisationswere performed using this two-layer approach.

While the 60 min films have been grown for 12 times longerthan the 5 min films the amounts of incorporated NT-3 is not 12times greater. The data presented in Fig. 4 clearly shows a linear

increase in NT-3 loading with polymerisation time; however itis clear that the linear regression lines do not intercept the y-axisthrough the zero point. This suggests that processes occurring inthe very early stages of polymerisation influence the NT-3loading. Upon investigation of the resulting voltage generatedduring polymerisation, it was observed that the voltage valuewas consistently higher when NT-3 was present in thepolymerisation solution. The chronopotentiograms (voltageverses time) recorded during polymerisation in the presenceof NT-3 also showed variation in the initial stages (first 30 s) ofpolymerisation compared to polymerisation with no NT-3present. When NT-3 was present in the monomer solution thevoltage drawn immediately upon commencement of polymer-isation was consistently higher compared to that observed in theabsence of NT-3. There is also the possibility of adsorption ofNT-3, either immediately after the PPy/pTS grown on gold-coated Mylar is placed into the monomer solution containingNT-3, or when the constant current is applied to the sample.

When the intercept values of the linear regression in Fig. 4are subtracted from the experimentally obtained data theamount of incorporated NT-3 at 60 min is, (within the statederrors), 12 times that of the 5 min grown polymer. Theseintercept values represent the amount of NT-3 incorporated intothe polymer films in the first 30 second polymerisation period,or shortly thereafter. To investigate further whether NT-3adsorption occurs at the PPy/pTS electrode surface, a PPy/pTSpolymer was placed into a solution of 1.35 ppm NT-3 thenimmediately removed (20–30 s) and rinsed thoroughly under astream of Milli-Q water. Gamma radiation counting showed that8.7 ng/cm2 of NT-3 was adsorbed.

The amount of NT-3 loaded into the polymers using the1.35 ppm was approximately 27 ng and 130 ng at 10 minute and60 minute growth respectively and was in excess of what istheoretically required to maintain neuron survival (this isespecially true in the cochlea where the volume is very smalland clearance is slow). Malgrange et al. [41] showed that invitro, 10 ng/ml in 100 μL volume (corresponding to 1 ng NT-3)is sufficient for nerve survival.

Fig. 5. Amount of NT-3 incorporated into (A) thin (3.6 μm) and (B) thick(26 μm) PPy/pTS films grown using a 2-layer approach (see text) at 2 mA/cm2

from monomer solution containing 0.2 M pyrrole, 0.05 M pTS and 1.35 ppmNT-3. The data show the average of 40 samples and the error bars represent onestandard error of the mean. The time in brackets indicates the polymerisationtime. The error bars represent the standard error.

290 B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

3.4. Incorporation of NT-3 into PPy/pTS films using optimalsynthesis conditions

The amount of NT-3 incorporated into PPy/pTS films wasdetermined by gamma emission of the 125I labelled NT-3incorporated into PPy/pTS films. The average of 10 minute and60 minute grown polymers were calculated across 40 sampleseach, and the amount of NT-3 within the films is shown inFig. 5. The values varied slightly between batches of radio-labelled NT-3, but are representative of the values obtained forall polymerisations using the two-layer approach with NT-3 at1.35 ppm. The thickness of these films are shown in Fig. 2 withthe 10 minute grown film being 3.6 μm thick and the 60 minutegrown film having a thickness of 26 μm. The 10 minute grownfilm will hereon be referred to as the “thin” film and the60 minute grown film the “thick” film. It is this variation in filmthickness that gives rise to the difference in the incorporatedamount of NT-3 shown in Fig. 5.

The NT-3 molecule possesses a positive charge in thepolymerisation solution with the proposed mechanism for NT-3incorporation into the polymer illustrated in Scheme 2. Cyclicvoltammetry of a solution containing only NT-3 using a gold-coated Mylar electrode produced no oxidation and reductionfeature indicating the NT-3 not to be electroactive. The basis ofthe NT-3 incorporation is not fully understood but is thought toinvolve ionic interactions with dopant anions, hydrophobicinteractions with either dopant or pyrrole molecules of theforming film, or physical entrapment of the protein molecules. Itwas found that in the absence of the dopant anion (A−) in thepolymerisation solution it was not possible to detect any

Scheme 2. Mechanism for incorporation of NT-3 into polypyrrole. A− representsa dopant anion such as para-Toluene sulfonate.

incorporated NT-3 molecule. This suggests that the dopingprocess may be the dominant process involved in theincorporation of NT-3.

While the thick polymer film has been grown for 6 timeslonger than the thin polymer film the amount of incorporatedNT-3 is not 6 times greater. This phenomenon was alsoobserved in Fig. 4, with interaction of NT-3 with thepolymerisation process and adsorption of NT-3 in the earlystages of polymerisation proposed as the cause.

3.5. Release of NT-3 from PPy/pTS/NT-3 by electricalstimulation in 0.9% NaCl

Electrochemically controlled release of NT-3 from identicalPPy/pTS/NT-3 polymers was performed by pulsed potential(±0.6 V at 5 Hz), cyclic voltammetry (±0.8 V to −1.0 V at50 mV/s) and pulsed current (±0.5 mA and ±20 mA at 5 Hz)(Fig. 6). The amount of NT-3 released after 12 min and 60 minof stimulation are shown.

Overall, the thick polymer films released a higher mass ofNT-3 than the thin polymer films, due to the higher loading(Fig. 6). However, the percentage of total incorporated NT-3

Fig. 6. Mass of NT-3 released from a (A) thin (3.6 μm) and (B) thick (26 μm)PPy/pTS/NT-3 polymer films in 0.9% NaCl (1 mL volume) by various electro-chemical release methods. Each point represents the average of 2–3 measure-ments, and the % values presented represent the average percentage of totalincorporated NT-3 release from the films. The 12 minute data represents theamount of NT-3 released after 12 min while the 60 minute data represents theamount released after 60 min (12 min release plus next 48 min release data). Theerror bars represent the standard error of each data set.

Fig. 7. Cumulative NT-3 release from (○) stimulated (pulsed current ±0.5 mA/cm2 at 5 Hz) and (O) unstimulated PPy/pTS/NT-3 films over 1 week from (A)thin (3.6 μm) and (B) thick (26 μm) polymer films. The polymer films weregrown using a current density of 2 mA/cm2. The release value representscumulative amounts of NT-3 release. The error bars represent the standard error.

291B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

released was much lower for the thick polymer films,suggesting that there may be some NT-3 physically trappedinside these films. Comparing the amounts of NT-3 releasedacross various stimulation protocols used in this study, cyclicvoltammetry (CV) and pulsed current (±0.5 mA) resulted inmore NT-3 release for the thin polymer films, while CV andpulse potential resulted in more NT-3 release for the thickpolymer films.

All release protocols resulted in greater amounts of NT-3being released in the first 12 min of stimulation compared to thenext 48 minute release (difference between the 10 minute and60 minute data). This suggests that these protocols may be moreeffective at releasing the NT-3 that is near the surface of thepolymer. This is especially true for the thick polymer samples.The total CV release reported represents only the 12 minutesample release, as the polymers delaminated from the electrodesurface with prolonged CV stimulation. SEMs obtained,following controlled release of NT-3, indicated that no crackingor delamination of the underlying gold-coated Mylar wasevident. The typical cauliflower morphology of the polymerwas retained (Fig. 3B) after 60 min controlled release using allof the stated release protocols (except for CV release wheredelamination occurred after 12 min). The electrical integrity wasalso confirmed with the CV recorded after release beingidentical to the CV presented in Fig. 1.

The mechanism for NT-3 release is proposed to involveelectrostatic interactions as the application of electricalstimulation protocol increases the amount of NT-3 release.However, both the ionic and hydrophobic properties ofpolypyrrole have been shown to vary upon application ofelectrical stimulus [36]. The expansion and contraction(actuation) [42,43] of polypyrrole is also a well documentedproperty of polypyrrole upon oxidation and reduction and theseprocesses may be involved in the release of NT-3. As theseproperties change simultaneously it is not possible to separatethem to determine which process is the driving force for release.

We have confirmed the bioactivity of the released NT-3through primary auditory nerve cell culture studies. Briefly,primary auditory nerve cells extracted from 5 day old rat pupscultured in media containing NT-3 released from thepolypyrrole showed the same extent of neural sprouting andgrowth as the auditory nerves cultured in media containing NT-3 that was not incorporated and released from polypyrrole. Theextent of auditory nerve sprouting and growth for both NT-3systems was greater than when the nerve cells were culturedwithout NT-3 present in the culture media. These findings arereported in more detail in an accompanying manuscript [47].

Other molecules such as small polypyrrole oligomers andpTS may also be released with the NT-3. Auditory nerve cellcultures were performed in the presence of trace amounts ofpTS and pyrrole (pyrrole diluted between 1:100 and 1:10,000and 0.05–50 mM pTS) to determine the possible toxicity ofthese other molecules. The pTS and pyrrole concentrations weredetermined based on the monomer concentrations and thepercentage of NT-3 release recorded. The cell culture studiesclearly showed healthy nerve cells with nerve sprouting evidentin the presence of these molecules. There was no difference in

the nerve cells phenotype and growth processes in the presenceof pyrrole and pTS compared to the same auditory nerve celltype cultured without pyrrole and pTS being present. It wasdetermined that at the concentrations used these molecules werenot cytotoxic to the auditory nerve cells used in this study.

3.6. Long-term release of NT-3 from PPy/pTS/NT-3 byelectrical stimulation in 0.9% NaCl

The effect of stimulation on the release of NT-3 from both thethin and thick PPy/pTS/NT-3 polymer films was consideredusing the pulsed current protocol of ±0.5 mA/cm2 at 5 Hz. Thisprotocol was chosen as it is characteristic of a protocol that isconductive to cell survival due to the low current density and thecharge balanced biphasic nature of the stimulation. Thick andthin polymer film samples were stimulated for a week with therelease media being collected and replaced at various timepointsthroughout. The cumulative release profile over the week can beseen compared to the release profile of an identical unstimulatedpolypyrrole sample in Fig. 7. The NT-3 amounts presented weredetermined by measuring the radiation of the receiving buffersolution removed from the controlled release cell. Theunstimulated and stimulated NT-3 release values are the average

292 B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

of triplicate experiments. In all experiments, electrical stimu-lation produced greater NT-3 release amounts.

For the thin polymer films (Fig. 7A) the stimulated polymerreleased more NT-3 than the unstimulated polymer, 5.6 ng/cm2

as opposed to 3.4 ng/cm2 respectively. The release profiles arecharacterised by a burst of NT-3 release in the first 24 hfollowed by a much lower amount over the next 6 days. Theamount of NT-3 released in the first 24 h constitutes 85% and95% of the total measured NT-3 release over the 7 days for thestimulated and unstimulated thin polymer films respectively.The same release trends are observed for the thick polymerfilms (Fig. 7B) with the stimulated polymer releasing more NT-3 than the unstimulated polymer, 8.8 ng/cm2 and 5.3 ng/cm2

respectively over the 7 days. The release profile of the thickpolymer film is similar to the thin polymer film and is alsocharacterised by a burst release in the first 24 h followed by amuch lower release over the next 6 days. The amount of NT-3released in the first 24 h constitutes 72% and 68% of the totalmeasured NT-3 release over the 7 days for the stimulated andunstimulated polymers respectively. These percentage valuesare lower than the thin polymer films suggesting these thickerfilms are able to further regulate the extent of release. It wasobserved for the unstimulated sample that upon activation ofelectrical stimulation at day 7 a rapid increase in NT-3 release

Fig. 8. Long-term (days 3 to 7) cumulative NT-3 release from (○) stimulated(pulsed current ±0.5 mA/cm2 at 5 Hz) and (O) unstimulated PPy/pTS/NT-3films over 1 week from (A) thin (3.6 μm) and (B) thick (26 μm) polymer films.The polymer films were grown using a current density of 2 mA/cm2. The releasevalue represents cumulative amounts of NT-3 release. The error bars representthe standard error.

was observed (data not shown). This phenomenon suggests thatthe release of NT-3 is not due to desorption of adsorbed NT-3from the polypyrrole surface. This aspect of the work iscurrently being investigated further.

The effect of stimulation on the later stages of long-termrelease (days 3 to 7) for both the thin (Fig. 8A) and thick(Fig. 8B) polymer films was further investigated. The linearregression of the data in Fig. 8A indicates in these later stage ofrelease stimulation at ±0.5 mA/cm2 at 5 Hz can release NT-3 at arate 3.7 times greater than unstimulated; 0.16 ng/day as opposedto 0.043 ng/day. The linear regression of the thick polymershowed the release rate under stimulation to be 3.5 times greaterthan unstimulated; 0.41 ng/day as opposed to 0.12 ng/day. Therate of NT-3 release from the thick polymer under both stimu-lated and unstimulated conditions was greater than from the thinpolymer. Considering the thick polymer contains more NT-3 it isto be expected to release larger amounts of NT-3 (Fig. 8B).

The release rates from both the thin and thick polymer filmsare below NT-3 delivery rates used for in vivo studies [31,44,].All current techniques for NT-3 delivery literally flood the entirecochlea with neurotrophins, with excessive amounts making itsway down the cochlear aqueduct and into other areas of thebrain resulting in significant amounts of the NT-3 beingconsumed by non-auditory cells. Wise et al. [44] showed thatadministering 10 μg NT-3 combined with 10 μg BDNF at a rateof 300 ng/day was sufficient for maintaining nerve survival andinitiating nerve resprouting in the deafened cochlea. While theamount and the rate of neurotrophin delivery used in their studywas much larger than the amount and release rate of NT-3described here, the researchers administered the neurotrophinsinto the cochlea at a site relatively distant to the neurons, andtherefore expected only a small proportion of the neurotrophinadministered to reach the nerve cells. Hence greater amounts,and an increased delivery rate, of NT-3 were used to ensureadequate diffusion to the damaged nerves.

Miller et al. [31] released NT-3 at a rate of 0.6 ng/day over14 days (for a total of 8.4 ng) into the cochlear fluids of theguinea pig cochlea via a mini-osmotic pump. This rate ofdelivery is between approximately 14 and 3.6 times greaterthan what was observed for the thin polymer withstimulated=0.16 ng/day or unstimulated=0.043 ng/day. Forthe thick polymer films the rate of release (stimulated=0.41 ng/day and unstimulated=0.12 ng/day) are a factor of 1.5 and 5 lessthan the Miller results. However, as was reported by Wise [44],the neurotrophin delivery used byMiller et al. [31] takes place ata location removed from the site of nerve damage. In contrast,the polymer neurotrophin delivery system described here isexpected to release the neurotrophin directly to the required site,ensuring that the majority of released NT-3 is available to betaken up by the damaged nerves.We propose that use of a releasedevice such as the one proposed here would allow the releaselocation to be directly at the site of the neurons and less NT-3release would be required. This is currently under investigationwith our preliminary findings recently reported [47].

The higher initial rate of NT-3 released (Fig. 7) may provide alarge amount of NT-3 to diffuse away from the source (polymer),and produce a concentration gradient. Nerve cells grow toward

293B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

the source of an NT-3 gradient, and so the higher initial valuesmay encourage neuron growth towards the polymer. The initialburst of neurotrophin is likely to be effective for several days, asthe signalling molecules are thought to be recycled several timesbefore retrograde transport [30]. After this burst, the rate ofrelease is much lower, which may help to promote signallingspecifically to cells in the local environment. In other words, theinitial burst may help to attract neurons to the site of release, andthe long-term slow release of NT-3 would help to promotesurvival of neurons by providing a small localised signal to theneurons at the site of NT-3 release. The small long-term signalmay be important, as high levels of NT-3 can cause Schwann cellhypertrophy, and NT-3 has been shown to down regulateexpression of trk receptors [45,46] and may even influencenociception [46]. However, a more linear release profile is moredesirable, and nanostructuring of the electrode surfaces is likelyto produce a better release profile (currently under investiga-tion). Another way to modulate NT-3 release may be to use othermolecules to dope the polypyrrole, which may also allow anti-bacterial, anti-fibroblastic or extracellular matrixmolecules to beincorporated into the polymer.

4. Conclusions

Polypyrrole doped with pTS and NT-3 has been shown toperform as a controlled release device. The incorporation of NT-3 into polypyrrole was optimised using galvanostatic polymer-isation at 2 mA/cm2 from a monomer solution containing 0.2 Mpyrrole, 0.05 M pTS and 1.35 ppm NT-3. The amount of NT-3incorporated into the polymer increased with extendedpolymerisation time. The controlled release of NT-3 wasinvestigated using a variety of electrochemical stimulationapproaches. While the polymer with the higher NT-3 loadingreleased greater amounts of NT-3 they released lowerpercentages of the total incorporated NT-3. Essentially, thepolymer grown for shorter periods was able to release a greaterpercentage of the incorporated NT-3. To maintain sufficient NT-3 release and polymer integrity it was necessary to employ amore cell “friendly” release protocol. All of the electrochemicalstimulation approaches released more NT-3 than unstimulated(natural diffusion) release. The long-term release profiles, bothstimulated and unstimulated, where characterised by a burstrelease occurring within the 24 h followed by a much slowerrelease over the next 6 days. The amount of incorporated andreleased NT-3 is proposed to be sufficient to regenerate andencourage the proliferation of damaged nerves.

Acknowledgments

BCT, SEM, JD, AC, GGW and GMC acknowledge thecontinued support of the Australian Research Council (ARC).Research conducted at the Bionic Ear Institute was generouslysupported by the Friends of Flint Hill Foundation and theStavros S. Niarchos Foundation. The authors also wish to thankDr. Alexey V. Pan (Institute for Semiconducting and ElectronicMaterials, University of Wollongong) for providing polymerthickness data.

References

[1] X.D. Wang, X.S. Gu, C.W. Yuan, S.J. Chen, P.Y. Zhang, T.Y. Zhang, J.Yao, F. Chen, G. Chen, Evaluation of biocompatibility of polypyrrole invitro and in vivo, J. Biomed. Mater. Res. 68A (2004) 411–422.

[2] N. Alikacem, Y. Marois, Z. Zhang, J.B. Jakubiec, R. Roy, M.W. King, R.Guidoin, Tissue reactions to polypyrrole-coated polyesters: a magneticresonance relaxometry study, Artif. Organs 23 (10) (1999) 910–919.

[3] B. Jakubiec, Y. Marois, Z. Zhang, R. Roy, M.F. Sigot-Luizard, F.J. Dugré,M.W. King, L. Dao, G. Laroche, R. Guidoin, In vitro cellular response topolypyrrole-coated woven polyester fabrics: potential benefits of electricalconductivity, J. Biomed. Mater. Res. 41 (1998) 519–526.

[4] Z. Zhang, R. Roy, F.J. Dugré, D. Tessier, L.H. Dao, In vitrobiocompatibility study of electrically conductive polypyrrole-coatedpolyester fabrics, J. Biomed. Mater. Res. 57 (2001) 63–71.

[5] S.J. Chen, C.W. Yuan, X.D. Wang, P.Y. Zhang, X.S. Gu, Research ofoutgrowth of polypyrrole and its biocompatibility with nervous tissue,Prog. Biochem. Biophys. 27 (2000) 212–214.

[6] J.H. Collier, J.P. Camp, T.W. Hudson, C.E. Schmidt, Synthesis andcharacterization of polypyrrole-hyaluronic acid composite biomaterials fortissue engineering applications, J. Biomed.Mater. Res. 50 (2000) 574–584.

[7] B. Garner, A. Georgevich, A.J. Hodgson, L. Liu, G.G. Wallace,Polypyrrole-heparin composites as stimulus-responsive substrates forendothelial cell growth, J. Biomed. Mater. Res. 44 (1999) 121–129.

[8] C. Lia, K.G. Neoh, Y.L. Li, E.T. Kang, Assessment of in vitro bioactivityof hyaluronic acid and sulfated hyaluronic acid functionalized electro-active polymer, Biomacromolecules 5 (2004) 2238–2246.

[9] C.E. Schmidt, V.R. Shastri, J.P. Vacanti, R. Langer, Stimulation of neuriteoutgrowth using an electrically conducting polymer, Proc. Natl. Acad. Sci.U. S. A. 94 (1997) 8948–8953.

[10] G.G. Wallace, L.A.P. Kane-Maguire, Manipulating and monitoringbiomolecular interactions with conducting electroactive polymers, Adv.Mat. 14 (2002) 953–960.

[11] M. Hepel, Composite polypyrrole films switchable between the anion- andcation-exchanger states, Electrochim. Acta 41 (1996) 63–76.

[12] M.R. Gandhi, P. Murray, G.M. Spinks, G.G. Wallace, Mechanism ofelectromechanical actuation in polypyrrole, Synth.Met. 73 (1995) 247–256.

[13] S.J. Taylor, E.S. Rosenzweig, J.W. McDonald III, S.E. Sakiyama-Elbert,Delivery of neurotrophin-3 from fibrin enhances neuronal fiber sproutingafter spinal cord injury, J. Control. Release 113 (2006) 226–235.

[14] M.L.T. Zweers, G.H.M. Engbers, D.W. Grijpma, J. Feijen, Release of anti-restenosis drugs from poly(ethylene oxide)-poly(DL-lactic-co-glycolicacid) nanoparticles, J. Control. Release 114 (2006) 317–324.

[15] H.K. Kim, H.J. Chung, T.G. Park, Biodegradable polymeric microsphereswith “open/closed” pores for sustained release of human growth hormone,J. Control. Release 112 (2006) 167–174.

[16] N.Csaba,A.Sánchez,M.J.Alonso, PLGA:Poloxamer andPLGA:Poloxamineblend nanostructures as carriers for nasal gene delivery, J. Control. Release 113(2006) 164–172.

[17] A. Aubert-Pouessel, M.C. Venier-Julienne, A. Clavreul, M. Sergent, C.Jollivet, C.N. Montero-Menei, E. Garcion, D.C. Bibby, P. Menei, J.P.Benoit, In vitro study of GDNF release from biodegradable PLGAmicrospheres, J. Control. Release 95 (2004) 463–475.

[18] Y. Zhong, R.V. Bellamkonda, Controlled release of anti-inflammatoryagent α-MSH from neural implants, J. Control. Release 106 (2005)309–318.

[19] R. Wadhwa, C.F. Lagenaur, X.T. Cui, Electrochemically controlled releaseof dexamethasone from conducting polymer polypyrrole coated electrode,J. Control. Release 110 (2006) 531–541.

[20] X. Cui, V.A. Lee, Y. Raphael, J.A. Wiler, J.F. Hetke, D.J. Anderson, D.C.Martin, Surface modification of neural recording electrodes in conductingpolymer/biomolecule blends, J. Biomed. Mater. Res. 56 (2001) 261–272.

[21] X. Cui, D.C. Martin, Fuzzy gold electrodes for lowering the impedanceand improving adhesion with electrodeposited conducting polymer films,Sens. Actuators, A, Phys. 103 (2003) 384–394.

[22] X. Cui, J.F. Hetke, J.A.Wiler, D.J. Anderson, D.C.Martin, Electrochemicaldeposition and characterisation of conducting polymer polypyrrole?PSS onmultichannel neural probes, Sens. Actuators, A, Phys. 93 (2001) 8–18.

294 B.C. Thompson et al. / Journal of Controlled Release 116 (2006) 285–294

[23] P. Ernfors, M.L. Duan, W.M. ElShamy, B. Canlon, Protection of auditoryneurons from aminoglycoside toxicity by neurotrophin-3, Nat. Med. 2(1996) 463–467.

[24] D.R. Kaplan, F.D. Miller, Signal transduction by the neurotrophinreceptors, Curr. Opin. Cell. Biol. 9 (1997) 213–221.

[25] R. Levi-Montalcini, S.D. Skaper, R. Dal Toso, L. Petrelli, A. Leon, Nervegrowth factor: from neurotrophin to neurokine, Trends Neurosci. 19 (1996)514–520.

[26] M. Ryden, R. Sehgal, C. Dominici, F.H. Schilling, C.F. Ibanez, P. Kogner,Expression of mRNA for the neurotrophin receptor trkC in neuroblastomaswith favourable tumour stage and good prognosis, Br. J. Cancer 74 (1996)773–779.

[27] E.C. Yuan, W.C. Mobley, Therapeutic potential of neurotrophic factor forneurological disorders, Ann. Neurol. 40 (1996) 346–354.

[28] P.L. Marzella, G.M. Clark, R.K. Shepherd, P.F. Bartlett, T. Kilpatrick,Synergy between TGF-beta 3 and NT-3 to promote the survival of spiralganglia neurones in vitro, Neurosci. Lett. 240 (1998) 77–80.

[29] M. Bothwell, Functional interactions of neurotrophins and neurotrophinreceptors, Ann. Rev. Neurosci. 18 (1995) 223–253.

[30] M.W. Weible II, S.E. Bartlett, A.J. Reynolds, I.A. Hendry, Prolongedrecycling of internalized neurotrophins in the nerve terminal, Cytometry 43(2001) 182–188.

[31] J.M. Miller, D.H. Chi, L.J. Okeeffe, P. Kruszka, Y. Raphael, R.A.Altschuler, Neurotrophins can enhance spiral ganglion cell survival afterinner hair cell loss, Int. J. Devl. Neurosci. 15 (1997) 631–643.

[32] R.B. Borgens, Electrically mediated regeneration and guidance of adultmammalian spinal axons into polymeric channels, Neuroscience 91 (1999)251–264.

[33] A. Kotwal, C.E. Schmidt, Electrical stimulation alters protein adsorptionand nerve cell interactions with electrically conducting biomaterials,Biomaterials 22 (2001) 1055–1064.

[34] W.M. Saltzman, W.L. Olbricht, Building drug delivery into tissueengineering, Nat. Rev. Drug Discov. 3 (2002) 177–186.

[35] B.L. Seal, T.C. Otero, A. Panitch, Polymeric biomaterials for tissue andorgan regeneration, Mater. Sci. Eng., R Rep. 34 (2001) 147–230.

[36] G.G. Wallace, G.M. Spinks, L.A.P. Kane-Maguire, P.R. Teasdale,Conductive Electroactive Polymers: Intelligent Material Systems, SecondeditionCRC Press, Boca Raton, 2003 and references cited therein.

[37] H. Zhao, W.E. Price, C.O. Too, G.G. Wallace, Parameters influencingtransport across conducting electroactive polymer membranes, J. Membr.Sci. 119 (1996) 199–212.

[38] H. Zhao, W.E. Price, G.G. Wallace, Synthesis, characterisation andtransport properties of layered conducting electroactive polypyrrolemembranes, J. Membr. Sci. 148 (1998) 161–172.

[39] T.W. Lewis, G.M. Spinks, G.G. Wallace, A. Mazzoldi, D. De Rossi,Investigation of the applied potential limits for polypyrrole whenemployed as the active components of a two-electrode device, Synth.Met. 122 (2001) 379–385.

[40] S.E. Moulton, J.N. Barisci, A. Bath, R. Stella, G.G. Wallace, Investigation ofprotein adsorption and electrochemical behaviour at a gold electrode, J. ColloidInterface Sci. 261 (2003) 312–319.

[41] B. Malgrange, P. Lefebvre, T.R. Van de Water, H. Staecker, G. Moonen,Effects of neurotrophins on early auditory neurones in cell culture,NeuroReport 7 (1996) 913–917.

[42] A.S. Hutchison, T.W. Lewis, S.E. Moulton, G.M. Spinks, G.G. Wallace,Development of polypyrrole-based electromechanical actuators, Synth.Met. 113 (121) (2000) 2000.

[43] T.W. Lewis, S.E. Moulton, G.M. Spinks, G.G. Wallace, Synth. Met 85(1997) 1419–1420.

[44] A. Wise, R.T. Richardson, J. Hardman, G.M. Clark, S. O'Leary,Resprouting and survival of guinea pig cochlear neurons in response tothe administration of the neurotrophins brain-derived neurotrophic factorand neurotrophin-3, J. Comp. Neurol. 487 (2005) 147–165.

[45] S. Wyatt, G. Middleton, E. Doxakis, A.M. Davies, Selective regulation of trkCexpression byNT3 in the developing peripheral nervous system, J.Neurosci. 19(1999) 6559–6570.

[46] K.A. Gratto, V.M.K. Verge, Neurotrophin-3 down-regulates trkA mRNA,NGF high-affinity binding sites, and associated phenotype in adult DRGneurons, Eur. J. Neurosci. (2003) 1535–1548.

[47] R.T. Richardson, B. Thompson, S. Moulton, C. Newbold, M.G. Lum, A.Cameron, G. Wallace, R. Kapsa, G. Clark, S. O’Leary, The effect ofpolypyrrole with incorporated neurotrophin-3 on the promotion of neuriteoutgrowth from auditory neurons, Biomaterials (in press).