poly(acrylic acid) interpenetrating polymer networks

17 Pages·2011·2.75 MB·English

by  Yi Lu

Biomaterials33(2012)378e394
ContentslistsavailableatSciVerseScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)-poly(vinyl alcohol)/
poly(acrylic acid) interpenetrating polymer networks for improving
optrode-neural tissue interface in optogenetics
Yi Lua, Yanling Lia, Jianqing Pana, Pengfei Weia, Nan Liua, Bifeng Wua, Jinbo Chenga,b, Caiyi Lub,
Liping Wanga,*
aShenzhenKeyLabofNeuropsychiatricModulation,ShenzhenInstitutesofAdvancedTechnology,ChineseAcademyofSciences,Shenzhen518055,China
bInstituteofGeriatricCardiology,ChinesePLAGeneralHospital,Beijing100853,China
a r t i c l e i n f o a b s t r a c t
Articlehistory: Thefieldofoptogeneticshasbeensuccessfullyusedtounderstandthemechanismsofneuropsychiatric
Received12September2011 diseases through the precise spatial and temporal control of specific groups of neurons in a neural
Accepted28September2011 circuitry.However,itremainsagreatchallengetointegrateoptogeneticmodulationwithelectrophysi-
Availableonline20October2011
ological and behavioral read out methods as a means to explore the causal, temporally precise, and
behaviorallyrelevantinteractionsofneuronsinthespecificcircuitsoffreelybehavinganimals.Inthis
Keywords: study, an eight-channel chronically implantable optrode array was fabricated and modified with
Optogenetics
poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)-poly(vinyl alcohol)/poly(acrylic acid) inter-
Optrode
penetrating polymer networks (PEDOT/PSS-PVA/PAA IPNs) for improving the optrode-neural tissue
Channelrhodopsin-2(ChR2)
Conductingpolymers(CPs) interface.Theconductingpolymer-hydrogelIPNfilmsexhibitedasignificantlyhighercapacitanceand
Hydrogel lower electrochemical impedance at 1 kHz as compared to unmodified optrode sites and showed
Neuralinterface significantlyimprovedmechanicalandelectrochemicalstabilityascomparedtopureconductingpoly-
merfilms.Thecellattachmentandneuriteoutgrowthofratpheochromocytoma(PC12)cellsontheIPN
films were clearly observed through calcein-AM staining. Furthermore, the optrode arrays were
chronicallyimplantedintothehippocampusofSDratsafterthelentiviralexpressionofsynapsin-ChR2-
EYFP,andlight-evoked,frequency-dependantactionpotentialswereobtainedinfreelymovinganimals.
Theelectricalrecordingresultssuggestedthatthemodifiedoptrodearraysshowedsignificantlyreduced
impedance and RMS noise and an improved SNR as compared to unmodified sites, which may have
benefitedfrom the improved electrochemical performance and biocompatibility of the deposited IPN
films.Allthesecharacteristicsaregreatlydesiredinoptogeneticapplications,andthefabricationmethod
ofconductingpolymer-hydrogelIPNscanbeeasilyintegratedwithothermodificationmethodstobuild
amoreadvancedoptrode-neuraltissueinterface.
(cid:1)2011ElsevierLtd.Allrightsreserved.
1. Introduction gain-of-function analyses of neural circuitry in behaving animals
andhasestablishedahighspatial-temporalresolutionmethodfor
Optogeneticsisatechnologythatcombinesopticalcontroland studyingneuralfunctionsandunderstandingthecharacteristicsof
genetic targeting using cell-type specific and optically-sensitive a neuronal circuit [3,4]. This technology has offered significant
proteins (e.g., channelrhodopsin-2 (ChR2) for excitation or hal- advantages andshowngreatpotential inbothunderstandingthe
orhodopsin(NpHR)forinhabitation)fortheprecisemanipulation mechanisms of neuropsychiatric diseases such as Parkinson’s
of neuronal functions [1,2]. Recent advances in optogenetics disease[5,6],anxiety[7],schizophrenia[8],epilepsy[9],depression
methodology has allowed precisely timed loss-of-function and [10],addiction[11],sleepdisorders[12],andsocialdysfunction[13]
andindevelopingnewtherapeuticstrategies.
A major advantage of optogenetics is that it enables simulta-
* Correspondingauthor.KeyLabofHealthInformaticsofChineseAcademyof neous electrophysiological read out and optical stimulation with
Sciences, Shenzhen Institutes of Advanced Technology, Chinese Academy of a millisecond precision in a cell-specific manner [14]. Unlike
Sciences,1068 Xueyuan Boulevard, University Town of Shenzhen, Xili Nanshan,
presentneuralmodulationtechnologiessuchasdeepbrainstim-
Shenzhen518055,China.Tel.:þ8675586392218;fax:þ8675586382233.
E-mailaddress:[email protected](L.Wang). ulation (DBS), optogenetics allows the excitation or inhibition of
0142-9612/$eseefrontmatter(cid:1)2011ElsevierLtd.Allrightsreserved.
doi:10.1016/j.biomaterials.2011.09.083
Y.Luetal./Biomaterials33(2012)378e394 379
complete populationsofspecificneuronalsubtypesandtheelec- electrode-neural tissue interface and reduces the quality of the
trophysiologicalrecordingoflocalneuronalactivityinvivousingan recordingsignals[26].
integratedopticalfiber-electrode(optrode).Inaddition,itprevents Manyattemptshavebeenmadetodevelopelectrodematerials
theformationofelectricalstimulusartifactsthatmayoverlapand to improve the electrode-neural tissue interface and restore the
influencethedataanalysisoftherecordingresults[1]. function of implanted neural electrodes. The application of con-
Toinvestigatethecomplexbrainprocessingmechanismsatthe ducting polymers (CPs) such as polypyrrole (PPy) and poly(3,4-
functionallevel,researcherscandirectlymeasureandmanipulate ethylenedioxythiophene) (PEDOT), which have been widely used
the activities of specific neurons or neural circuits in living, in many fields includingenergystorage[31] and biosensors [32],
behavinganimalsusingoptogenetics.Inmanycases,tounderstand hasnowextendedintothefieldofneuroscience[33].BothPPyand
theintrinsicneuralbasisofcognitiveandbehavioralphenomena, PEDOT exhibit particularly low electrochemical impedance and
weneedtocarryoutlong-termstudiesonthecharacteristicsofthe tissue-friendlycharacteristicsinvivo,whicharehighlydesiredfor
neural circuits that control animal behavior. Therefore, the ideal implantable neural electrodes [21,34,35]. However, serious
optrodes for optogenetics are chronically implantable arrays that delaminationshavebeenobservedonconductingpolymercoated
integrateopticalmodulationwithelectrophysiologicalandbehav- electrodes[29,36],anditisobviousthattheirelectrochemicaland
ioral read out methods and can be used to explore the causal, mechanicalstabilitiesneedtobefurtherimproved[33].
temporally precise, and behaviorally relevant interactions of Inourpreviouswork,weshowedthathydrogelcoatingssuchas
neurons in specific circuits in freely behaving animals [15,16]. poly(vinyl alcohol)/poly(acrylic acid) interpenetrating networks
However,mostofthecurrentlyusedoptrodesaretypicallymadeby (PVA/PAAIPNs)canimprovetheelectrode-neuralinterfaceandare
coupling a multi-mode optical fiber with a tungsten recording highlystableafterchronicimplantation[30].Onthebasisofthese
microelectrode[5,14,17e19]orbyintegratinganopticalwaveguide results, we investigated whether a combination of conducting
with amulti-electroderecordingarray [6,20].These optrodesare polymerandhydrogelswillmakealowimpedanceandbiostable
generallyusedinacutestudiesonanesthetizedrats.However,the interfaceforoptrodearrays.TailoredPVA/PAAIPNswithanappro-
development of chronically implantable optrodes for awake and priateswellingratioandbulkionicconductivityweresynthesized
freelymovinganimalsremainsagreatchallenge. and deposited on our custom-made implantable optrode arrays.
Anotherchallengeistodevelopadvancedoptrode-neuraltissue PEDOT/PSSwasthenelectrochemicaldepositedintoaswollenPVA/
interfacesforresearchonbrainfunctionanddestructionofneural PAAIPNfilm,andinterpenetratingPEDOT/PSS-PVA/PAAnetworks
circuitries. The primaryrequirements for these interfaces include were formed. Electrochemical characteristics such as cyclic vol-
consistent optogenetic modulation and electrophysiological tammetry(CV),electrochemicalimpedancespectrum(EIS),andthe
recordingfromaspecificgroupofneuronsattherequisitespatial- electrochemical and mechanical stability of PEDOT/PSS-PVA/PAA
temporalresolutionsoveralongperiodoftime. IPNs were evaluated. The chemical composition and surface
The electrode is a key element in the read out function of morphologyofthecompositeIPNfilmsweredeterminedbyRaman
implantable optrodes, because it determines the correlation microscopy and scanning electron microscopy, respectively. In
between the target neurons and the signal recording quality addition, the differentiation and neurite growth of PC12 cells on
requiredforoptogenetics.Inordertoimprovethespatialresolution PEDOT/PSS-PVA/PAA IPNs deposited on indium tin oxide (ITO)
ofrecordingmedia,high-densitymicro-scaleelectrodearrayshave surfaceswereinvestigatedbycalcein-AMstaining.Afterlentiviral
beendeveloped,whicharebiologicallytransparentandcanmini- expression(pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE),theIPN-
mizeinsertiontrauma[21].However,withadecreaseinsize,the coatedoptrodearrayswereimplantedintothehippocampusofSD
electrochemical impedance of electrodes has increased signifi- ratsfor4weeks.Optogeneticmodulationandelectrophysiological
cantly.Becauseelectrodeimpedanceisproportionaltothethermal recordingweresuccessfullycarriedoutonfreelymovingrats,and
noise and signal loss through shunt pathways, the signal quality theperformancesofthemodifiedandunmodifiedoptrodearrays
maydecreasewiththesizeoftherecordingelectrodes[22]. wereanalyzedintermofthesignalnoiseratio(SNR)androotmean
One of the most commonly used strategies fordecreasing the square (RMS) noise voltage of the action potentials, local field
electrochemicalimpedanceofmicroelectrodesinvolvesreversible potential (LFP), and electrochemical impedance in vivo. All these
chargetransferviatransductionbetweentheionicallyconducting resultswereevaluatedanddiscussedagainsttherequirementsof
tissueandelectronicallyconductingrecordingelectrode(typically, optogeneticapplications.
gold, platinum, or tungsten) bya double-layer capacitive mecha-
nism, a reversible Faradaic mechanism, or both [21,23]. One 2. Experimental
candidate electrode material is iridium oxide (IrO2), which is
widely used for both recording and stimulating microelectrodes, 2.1. Preparationofsamplesandoptrodes
owing to its superior electrochemical characteristics such as low
impedance (at a typical frequency of 1 kHz) and high charge Ptmicroelectrodeswereusedaselectrodepositingsubstratesfor
injectiondensitylimits[24]. electrochemical tests and morphological studies, and they were
Unfortunately, reducing the electrochemical impedance alone fabricated by sealing a Pt wire (100 mm in diameter, 99.95%) in
does not meet the requirements of chronic recording in vivo, aglasscapillary.Indiumtinoxide(ITO)glassslides(10mminwidth
becauseanothermajorbottleneckhinderingthelong-termappli- and 10 mm in length, CSG Holding Co. Ltd., China) were used as
cationsofneuralelectrodeistheinconsistentperformancecaused electrodepositingsubstratesfortheRamanandcellculturestudies
bythetissueresponses[25e28].Ithasbeenreportedinanumber invitro.Implantableopticalfiber-electrode (optrode) arrays,each
ofstudiesthatafterimplantationinthecentralnervesystem(CNS), containingoneopticalchannelandeightelectricalchannels,were
inflammatory responses are produced at the electrode-neural fabricated from optical fibers (200 mm in diameter, NA ¼ 0.37,
tissue interface and subsequently lead to tissue encapsulation Thorlabs,USA)andformvar-coatednickelchromiumwires(36mm
around the electrode [29]. This highly resistive astroglial encap- in diameter, California fine wire, USA) using a custom-made
sulationresultsintheelectricalisolationoftheelectrodesfromthe optrode mold for in vivo studies. Eight microelectrodes in an
targetneurons byhindering the diffusion of ions at the interface optrodearraywerearrangedintwoparallelrows,eachcontaining
and leads to a loss of neurons near the interface [30]; this fourwires,andthespacingbetweenneighboringmicroelectrodes
dramatically increases the electrochemical impedance at the was200mm.Approximately2mmoftheinsulationwasremoved
380 Y.Luetal./Biomaterials33(2012)378e394
fromoneendofeachmicroelectrodeusingabriefflame,andeach whereL,S,andRarethethickness,area,andbulkresistanceofthe
microelectrode was soldered into separate slots of a standard films,respectively.Thebulkresistanceofthefilmswasdetermined
electrode connector. Two pairs of silver microwires (100 mm in from the high-frequency intercept on the real impedance axis of
diameter,99.95%)werethensolderedintotheelectrodeconnector aColeeColeplot.
as ground and reference electrodes, respectively. One end of the
opticalfiberswasfixedontoacustom-madeopticalconnectorand
stabilizedontotheelectrodeconnectorusingalayerofepoxy. 2.4. Electrodepositionprocess
Allsamplesfortheelectrochemicalstudieswereultrasonically
cleanedinacetoneandultrapurewaterthreetimespriortouse.All 3,4-Ethylenedioxythiophene (EDOT) and poly(sodium
samplesforthecellcultureandimplantationstudiesweresteril- 4-styrenesulfonate) (PSSNa, MW ¼ 70,000) were purchased from
izedusing75%alcoholandultraviolet(UV)radiationpriortouse. SigmaeAldrich, USA. In a standard preparation procedure, the
electrodepositionsubstrateswerecoatedwithathinPVA/PAAIPNs
2.2. SynthesisofPVA/PAAIPNs film and dried slowly at 80 (cid:2)C overnight in a vacuum oven. The
coated substrates were then immersed in an electrodeposition
Poly(vinylalcohol)(PVA,MW¼89,000e98,000)andacrylicacid solutionforatleast2hsothatswellingequilibriumwasattained
(AA) werepurchased fromSigmaeAldrich, USA. Ammoniumper- prior to use. In all electrochemical experiments, the electrodes
sulfate (AR) was purchased from Degussa-AJ, Shanghai, China. were mounted in a three-electrode cell with a saturated calomel
AqueousPVAsolution(2wt%)waspreparedbydissolvingPVAin electrode(SCE)asareferenceelectrodeandalarge-areaplatinum
ultrapure water (resistivity of 18.25 MU/cm) at 95 (cid:2)C under electrodeasacounterelectrode.
magnetic stirring. The solution was then cooled down to room A 0.01M EDOT monomer and 0.25M PSSNa were mixed to
temperature and transferred into a three-neck flask fitted with prepare an electrodeposition solution. PEDOT/PSS films were
acondenser.AnappropriateweightoftheAAmonomerwasadded electrodeposited onto PVA/PAA-IPN-hydrogel-coated microelec-
tothe PVA solution under magnetic stirring at the desired ratios trodesusingaCHInstrumentsCHI660Danalyzerat0.9V(vs.SCE).
(0,10,20,30,40,50,60,70,and80mol%ofAAmonomerpertotal TocomparethePEDOT/PSS-PVA/PAAIPNfilmswithpureCPsfilms,
repeating unit). Ammonium persulfate was added to the flask at PEDOT/PSS films wereelectrodeposited onto uncoated substrates
1000 ppm as an initiator. The mixed solution was purged with under optimized conditions. PEDOT/PSS-PVA/PAA IPN films were
argon and stirred for 15 min. The flask was then sealed and alsocoatedonITOglassslidesforRamanandcellculturestudies
immersed in an oil bath at 80 (cid:2)C. The reaction was allowed to usingthesameprocedureasdescribedabove.
proceedfor72h.Theresultingpolymersolutionwasfilteredand
keptovernighttoremovetheundissolvedsolidsandbubblesprior
touse.ThehomogeneouspolymersolutionwaspouredontoaPTFE 2.5. ChemicalcharacterizationofPEDOT/PSS-PVA/PAAIPNs
platetoformafilmforchemicalcharacterizations.Theexcesswater
wasevaporatedslowlyat80(cid:2)Cfor12hinavacuumoven. 2.5.1. Ramantest
The prepared polymer films were characterized by measuring
2.3. ChemicalcharacterizationofPVA/PAAIPNs micro-Ramanspectra.TheanalysiswasconductedusingaRaman
Renishaw RM1000 micro-spectrometer (Renishaw plc, Wotton-
2.3.1. Swellingexperiments under-Edge, Gloucestershire, UK) with an excitation wavelength
Theswellingratiosofpolymerfilmswereevaluatedinbothpure of514 nm and a spectral resolution of 1 cm(cid:3)1. The micro-Raman
water and an artificial cerebrospinal fluid (ACSF, pHw7.4). Dry spectraweremeasuredwithintherangeof4000e400cm(cid:3)1.
polymerfilmswereweighedandthen immersedintothe testing
liquidfor48htoreachswellingequilibriumatroomtemperature. 2.5.2. Electrochemicalmeasurements
The free liquid on the surface of the swollen films was then Cyclicvoltammetric(CV)measurementswereperformedin0.1M
removedquicklyusingfilterpaper,andtheweightsofthesamples phosphate-bufferedsaline(PBS,pH7.3)between(cid:3)0.6Vand0.6V
weremeasuredagain.Theswellingratio(SR)ofthefilmsisdefined (vs.SCE)atascanrateof50mV/susingaCHInstrumentsCHI660D
asfollows: analyzerat25(cid:2)C.Thecathodicchargestoragecapacity(CSCC)was
calculated from the time integral of the cathodic current in one
SR ¼ ðmt(cid:3)moÞ=mo(cid:4)100%; (1) periodoftheCVwaveform.Theelectrochemicalimpedancespec-
where mo and mt are the weight of dry and swollen films, tinruAmCS(FEaISt)2o5f(cid:2)tChueseinlegctthroedCeHpIo6si6t0eDdamniaclryozeelrecwtritohdeas10w-amsVm(ermassu)rAeCd
respectively.
sinusoidsignalinafrequencyrangeof100kHzto1Hzattheopen
circuitpotential(vs.SCE).
2.3.2. Bulkionicconductivity
Thebulkionicconductivityofpolymerfilmswastestedinboth
pure water and ACSF. Dry polymer films were immersed in the 2.5.3. Stabilitytest
ThestabilityofthePEDOT/PSS-andPEDOT/PSS-PVA/PAA-IPN-
testingliquidandmaintainedatroomtemperaturefor48h.Then,
thefreeliquidonthesurfaceofthefilmswasremoved.Theswollen deposited microelectrodes was examined in ACSF by continuous
hydrogelfilmswerethencutintodiskswiththesamesizeasthe cyclicscanningbetweenthepotentialsof(cid:3)0.6Vand0.6V(vs.SCE)
testelectrodes.Thepolymerfilmswerethensandwichedbetween atascanrateof50mV/sfor100cycles.Then,themicroelectrodes
wereimmersedintheACSFfor1week,afterwhichthevariationsin
two symmetrical stainless steel blocking electrodes (each
w1.77 cm2 in area), and the AC impedance was measured in the CSCCweremeasuredtoevaluatethestabilityofthedepositedfilms.
frequencyrangeof100kHzto100Hzwitha10-mV(rms)sinusoid
2.5.4. Morphology
signalusingaCHInstrumentsCHI660Delectrochemicalanalyzer.
Thebulkionicconductivity(s)ofthefilmswasdeterminedusing Thesurfacemorphologiesandmicrostructuresofthedeposited
polymer films were examined using an extreme high-resolution
thefollowingequation
scanning electron microscope (XHRSEM, FEI Magellan 400L, US)
s ¼ L=ðS(cid:4)RÞ; (2) operatedat5kV.
Y.Luetal./Biomaterials33(2012)378e394 381
2.6. PC12cellculture the subject was fixed in a stereotaxic apparatus (Stoelting, USA),
amid-saggitalincisionwasmadeatthescalp,anda1.0-mmhole
Ratpheochromocytoma(PC12)cellswereusedasanervecell wascarefullydrilledat4.2-mmposteriortothebregmaand3-mm
model system to investigate the neuronal differentiation and leftlateraltothemidline.Synapsin-ChR2-EYFPviruswasinjected
extensiononthedepositedITOglassslides.TheseedsofthePC12 intothelefthippocampus(1mlat(cid:3)3.0mmand(cid:3)4.0mmDVfrom
cells were obtained from the American Type Culture Collection the top of the brain) through a 33-gauge metal needle. A 5-ml
(ATCC) and cultured by the China Southern Cell Company Hamilton microsyringe was used to deliver a concentrated virus
(Guangzhou, China). In ordertofacilitate the attachmentof non- solution via a microsyringe pump (Sarasota, FL, USA) and its
adheringPC12cells,allthesampleswerecoatedwith0.1mg/mL controller(Micro4;WPI,Sarasota,FL,USA).
ofpoly-L-lysine(PLL,Sigma,SouthSanFrancisco,CA,USA)at37(cid:2)C
foratleast2handwerethenrinsedwithsterilewaterfollowedby 2.7.4. Virusexpression
0.01M PBS prior to use. The PC12 cells were maintained in RPMI The animals were sacrificed two weeks after the lentiviral
1640medium(Gibco,NY,USA)supplementedwitha10%fetalbo- injection, and 120 mm thickcoronal cortical slices wereprepared
vine serum (Gibco, NY, USA), 2 mM L-glutamine (Gibco, NY, US), using a vibratome (VT 1000; Leica) in ice cold ACSF. ChR2
100U/mlpenicillin,and100mg/mlstreptomycin(Sigma,SouthSan expressingneuronswereidentifiedusingEYFPfluorescenceunder
Francisco,CA,USA)ina37(cid:2)Cincubator(5%CO2).Thesampleswere aninvertedfluorescencemicroscope(LeicaDMI3000B,German),
fixed on each bottom of a 6-well plate, and the PC12 cells were andimageswereobtainedwithadigitalcamera(Leica,German).
seeded at a density of 5 (cid:4) 104 cells/mL in each well and then
subjectedto6-hserumstarvation.50ng/mLofNGF(Sigma,South 2.7.5. Implantationsurgery
SanFrancisco,CA,USA)wasaddedontheseconddayoftheculture Two weeks after the lentiviral injection, the animals were
toallowcelladhesionbeforeinducingdifferentiation. anaesthetizedwith1%phenobarbitalsodiumsolution(1ml/100g)
Toinvestigatetheneuritedifferentiationandoutgrowthofthe andplacedinastereotaxicapparatusforoptrodearrayimplanta-
PC12 cells on the deposited surfaces, the cells were labeled with tionandelectrophysiologicalrecording.Supplementaldoseswere
calcein-AMafterculturinginthepresenceofNGFfor7days.The giventomaintainasteadyanestheticstate.Thescalpwasopened,
samples were first washed with 0.1M PBS and then incubated in and a hole was drilled into the skull at 4.2-mm posterior to the
RPMI 1640 medium with 5 mM of calcein-AM (Sigma, South San bregmaand3-mmleftlateraltothemidline.Fourskullscrewswere
Francisco,CA,USA)for30minat37(cid:2)C.Aftercalcein-AMloading, drivenintotheskulltoserveasreferenceandgroundelectrodes.
the cells were washed with PBS, and fluorescence images were Four neighboring channelsof each fabricated optrodearraywere
observed and recorded using an Olympus X71 inverted fluores- modifiedusingPEDOT/PSS-PVA/PAAIPNsbytheabovementioned
cencemicroscope. standardprocesspriortouse.Anoptrodearraywasloweredinto
the hippocampus (3.5 mm below the surface of the brain) and
2.7. Implantationstudyinvivo cemented in place with dental acrylic (Ortho-Jet, Lang Dental).
Acuterecordingsweremadeimmediatelyafterthecementdried.
2.7.1. Animals The animals were allowed to recover for at least 7 days before
All theprocedures wereconductedusingsterile techniques in further recordings were taken. Analgesia was applied before
accordancewiththeprotocolsapprovedbytheEthicsCommittee surgeryandfor3daysaftersurgery.
forAnimalResearch,ShenzhenInstituteofAdvancedTechnology,
Chinese Academy of Sciences. Adult male SpragueeDawley (SD) 2.7.6. Opticalstimulationandelectricalrecording
ratsweighting180e220gwereusedforchronicimplantation.The A473-nmbluelasercontrolledbyananaloginputwasusedfor
SDratswereindividuallyhousedfor7daysbeforevirusinjection ChR2 activation. The laser was conducted into the implanted
undera12-hlight/darkcycleandprovidedwithfoodandwaterad optrodearrayusinga3-mlongopticalfiber(200mmindiameter,
libitum. NA¼0.37,Thorlabs,USA)terminatedwithacustom-madeoptical
connector.Beforeimplantation,thepowerofthelaseratthetipof
2.7.2. Viruspreparation theoptrodearraywasmeasuredusingapowermeter.Thepower
DNAplasmidencodingpLenti-hSyn-hChR2(H134R)-EYFP-WPRE densityofthebluelightwasw10mW/mm2,andthestimuliwere
wasobtainedfromthelaboratoryofK.Deisseroth(seehttp://www. 5-mplusesat10Hz.
optogenetics.org for additional details). The plasmid DNA was Recordingswereperformedusinga64-channelneuralacquisi-
amplified,purified,andcollectedusingastandardplasmidmega- tion processor (Plexon, Dallas, TX, USA). During the recording
prepkit(Qiagen,Germany).Hightiterlentivirus(>109pfu/ml)was sessions,apreamplifier(Plexon,Dallas,TX,USA)wasconnectedto
thenproducedviaFuGENEHD(Roche,Switzerland)co-transfection the output connector of the optrode. Neural electrophysiological
of293FTcellsusing22mgofalentiviralvector,15mgofpCMVDR8.74, data for all the eight recording channels were bandpass filtered.
and7mgofpMD2.G.24hpost-transfection,the293FTcellswere Briefly, single and multi-unit recordings weresampled at 40 kHz
transferred to an UltraCULTURE medium (LONZA: 12-725F) con- and bandpass filtered at 300e5000 Hz, and local field potentials
taining5mMsodiumbutyrate;thesupernatantwascollectedafter (LFP) weresampled at 1 kHz and bandpass filtered at 1e500 Hz.
48handconcentratedusingultracentrifugationat25,000rpm.The Multiple90-ssegmentsofcontinuousneuralrecordings(from30s
resulting viral pellet was resuspended in 100 ml of phosphate- before to 30 s after optical stimulation) were taken. Neural
bufferedsaline. recordings for each optrode array were taken every week after
implantation. The animals were freely moving during chronic
2.7.3. Virusinjection electrophysiologicalrecording.
pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE DNA was driven
byasynapsin1promoterandhousedboththeChR2andenhanced 2.7.7. OptrodeEISinvivo
yellowfluorescentprotein(EYFP)forimagingthetransfectedbrain TheEISofthefabricatedoptrodearrayweretestedusingtheCHI
tissue. Anaesthesia was induced using 1% phenobarbital sodium 660Dwitha25-mV(rms)ACsinusoidsignalinafrequencyrangeof
solution(1ml/100g),andthebodytemperaturewasmaintained 100kHzto1Hzattheopencircuitpotential.Priortoimplantation,
usingaheatingpad(CMA/150,CMAMicrodialysis,Sweden).After theEISweremeasuredinACSFwithanSCEasareferenceelectrode,
382 Y.Luetal./Biomaterials33(2012)378e394
andalarge-areaplatinumelectrodeasacounterelectrode.TheEIS 3.1.2. Bulkionicconductivity
of the implanted optrode array was obtained using two stainless The bulk ionic conductivity of different PVA/PAA IPN
steel bone screws serving as reference and counter electrodes, membranes(PAAcontent¼0,10,20,30,40,50,60,70,and80mol
respectively.TheEISforeachoptrodearraywastakeneachweek %)inpurewaterandACSFareshowninFig.2.Obviously,thebulk
afterimplantation,andtheanimalswereanesthetizedthroughout ionicconductionofPVA/PAAIPNmembraneinpurewaterismainly
thedatacollectionsessions. causedbythetransferofHþionswithinthemembrane,whichis
proportional to the concentration of unreacted carboxylic acid
2.7.8. Dataanalysis groupsinthehydrogel[30].Asaresult,thebulkionicconductivity
Offline neural signal analyses were performed using Plexon ofthepreparedmembraneshasincreasedfrom5(cid:4)10(cid:3)3mS/cmto
Offline Sorter software (Plexon Inc., Dallas, TX) and a custom- 1.2mS/cmwithanincreaseintheamountoffreecarboxylicacid
designedMatlab(MathworksInc.,MA)code.First,spikedetection groups.
and an offline sorting procedure were completed in the Plexon Unlikeinpurewater,thebulkionicconductivityofthePVA/PAA
Offline Sorter, as described by Nicolelis et al. [37]. All the sorted IPN membrane in ACSF rapidly increases with an increase in the
waveforms of the units were sent to the Matlab workspace, and PAAcontent,andreachesaplatformofw20mS/cm.Thismaybe
eachunitclusterwasstoredinamatrix,whereeachrowrepresents caused by two factors. Firstly, the swelling ratio of PVA/PAA IPN
a single waveform and each column represents the waveform membraneismuchhigherinACSFthaninwater,sotheamountof
samples.Inaddition,themeanwaveformofacertainunitcanbe ions within the membrane is also higher. Secondly, it has been
obtainedbyaveragingthematrixalongtherowvectors. reportedthatthebulkionicconductionofthemembraneinACSFis
Thesorteddatawereanalyzedusingthesignalnoiseratio(SNR), causedbyasegmentalmotionmechanismofthehydrogel[40],and
whichprovidesanevaluationmethodtocomparetheunitquality. therefore,ionictransferwithinthemembraneismucheasier.
SNRforagivenclusterwascalculatedusingthefollowingformula As an electrode coating material, especially for chronic neural
describedin[38]: electrophysiological recording and stimulation, a high swelling
ratio of the semi-cross-linked PVA/PAA IPN hydrogel benefits
SNR ¼ App=ð2(cid:4)SDεÞ; (3) higherionicmobilityandlowerelectrochemicalimpedanceatthe
electrode-neuralinterface,butthismayposeapotentialrisktoits
where App is the peak-to-peak voltage amplitude of the mean
waveformofeachunitandSDεisthestandarddeviationofnoiseε long-term stability. Therefore, there is a tradeoff between the
electrochemicalperformance(higherbulkionicconductivity)and
in a single unit. The amplitude, App, is the absolute value of the
mechanicalstability(lowerswellingratio).Takingallthesefactors
difference between the maximum and minimum voltages of the
into consideration, PVA/PAA IPN hydrogel with a PVA content of
mean waveform; the noise is obtained by subtracting the mean
waveform from each individual waveform, and noise ε is the 60%waschosenasthebestcandidatecoatingmaterialforfurther
studies.
collectionofallresidualvaluesirrespectiveoftheirpositioninthe
matrix.SDεisthestandarddeviationtakenoverallvaluesofε.
3.2. ChemicalpropertiesofPEDOT/PSS-PVA/PAAIPNs
3. Resultsanddiscussion 3.2.1. Ramantest
To study the molecular structure of conducting-polymer-
3.1. ChemicalcharacteristicsofPVA/PAAIPNs hydrogel composite films, the micro-Raman spectrum of the ITO
substrate and the PVA/PAA-, PEDOT/PSS-, and PEDOT/PSS-PVA/
3.1.1. Swellingproperties PAA-depositedfilmsweremeasured.TheRamanspectrumofthe
TheswellingratiosofPVA/PAAIPNmembraneswithdifferent PVA/PAAIPNfilm(Fig.3(B))showedthemaincharacteristicbands
PAA contents (0,10, 20, 30, 40, 50, 60, 70, and 80 mol%) in pure asfollows:842cm(cid:3)1attheCeCstretchingvibration,1100cm(cid:3)1at
waterand ACSFare shownin Fig.1. As can be seen, the swelling theCeCstretchingvibrationorCH2rocking,1336and1452cm(cid:3)1at
ratioofthePVA/PAAIPNmembranesinpurewaterdecreaseswith the CeH and OeH bending vibrations, 1700 cm(cid:3)1 at the C]O
anincreaseinPAAcontent,andreachesaminimumvalueinPVA/ stretching vibration, and 2915 cm(cid:3)1 at the CeH stretching vibra-
PAA IPN hydrogel with 20% PAA content. This suggests that the tion.TheRamanspectrumofPEDOT/PSS-depositedfilmsisshown
carboxylic acid groups in PAA may have partly reacted with the inFig.3(C).Thebandsat1249and1422cm(cid:3)1correspondedtothe
hydroxyl groups in PVA and that new intermolecular hydrogen Ca-Ca’ and symmetric Ca¼Cb(eO) stretching vibrations, respec-
bonds or ester groups in the IPNs were formed [30]. As a conse- tively.Thebandsat1498,1526,and1567cm(cid:3)1correspondedtothe
quence, the semi-cross-linked PVA/PAA IPN hydrogel resulted in asymmetricCa¼Cbstretchingvibration.
amorecompactpolymernetwork.However,afurtherincreasein However, in the Raman spectrum of PEDOT/PSS-PVA/PAA
thePAAcontentledtoanincreaseintheswellingratio.Thiscanbe composite films (shown in Fig. 3(C)), the band at 1249 cm(cid:3)1 is
explainedbythefactthatthecarboxylicacidgroupsinPAAtendsto splitintotwobands,whichareshiftedupto1265and1285cm(cid:3)1,
form stronger hydrogen bonds with water than the hydroxyl respectively.Thebandat1425cm(cid:3)1issplitintotwobands,which
groupsinPVA. areshiftedupto1426and1442cm(cid:3)1.Theintensityofthebandsat
TheswellingratioofthePVA/PAAIPNmembranesintheACSFis 1498, 1526, and 1567 cm(cid:3)1 are significantly increased. These
significantly higher than in pure water. This implies that the upwardshifts maybeattributedtothehigherdopedstate ofthe
unreactedcarboxylicacidgroupsinthePVA/PAAIPNhydrogelhave PEDOT/PSS-PVA/PAA composite films as compared to the pure
reacted with the cations (Naþ, Kþ) in the ACSF, and have partly PEDOT/PSS films [41,42]. Additionally, no bands for ITO are
dissociated the intermolecular hydrogen bonding of the semi- observed,butanewbandat1452cm(cid:3)1appears,overlappingwith
cross-linked hydrogel [39]. In addition, it should be noted that the main band at 1442 cm(cid:3)1, which maybe due tothe CeH and
theswellingratioofthemembraneincreasedatafasterratewhen OeHbendingvibrationofthePVA/PAAIPNhydrogel.Theseresults
thePAAcontentinthehydrogelwashigherthan50%.Thismaybe suggestthatthehighlydopedconductingpolymermaybeattrib-
aresultofasharpincreaseintheunreactedcarboxylicacidgroups utedtothehighconcentrationoffreecarboxylicacidgroupsinthe
andalesscompactpolymernetworkofthehydrogelathighPAA PVA/PAA IPN hydrogel. This also indicates that interpenetrating
contents. networks may be formed between the conducting polymers and
Y.Luetal./Biomaterials33(2012)378e394 383
Fig.1. SwellingratiosofPVA/PAAIPNmembranesinpurewaterandACSF(PAAcontent¼0,10,20,30,40,50,60,70,and80mol%).
hydrogel, which may significantly improve the stability of the filmsgrewfasterthanpureCPs,thecarboxylicacidgroupsinthe
depositedfilms. PVA/PAA hydrogel may have indeed anticipated in the electro-
depositingprocess.
3.2.2. Cyclicvoltammograms Theelectrochemicalcharacteristicsofthefabricatedelectrodes
Under the optimized conditions, the PEDOT/PSS films were werestudiedusingcyclicvoltammetricscanningbetween(cid:3)0.6V
deposited onto Pt microelectrodes and PVA/PAA IPN-coated Pt and0.6V(vs.SCE)atascanrateof50mV/s(showninFig.4).Ascan
microelectrodesatapotentialof0.9V(vs.SCE)for300s,andthe be seenfromthe cyclic voltammograms, no obvious oxidation or
totalappliedchargedensityduringelectrochemicaldepositionwas reductionpeakswereobservedforthePEDOT/PSS-modifiedelec-
1.4 C/cm2 and 2.2 C/cm2, respectively. Because the CP-hydrogel trode,butapairofredoxpeaksforthePEDOT/PSS-PVA/PAA-IPN-
Fig.2. BulkionicconductivityofPVA/PAAIPNmembranesinpurewaterandACSF(PAAcontent¼0,10,20,30,40,50,60,70,and80mol%).
384 Y.Luetal./Biomaterials33(2012)378e394
Fig.3. Micro-Ramanspectrumof(A)ITOsubstrateand(B)PVA/PAA-,(C)PEDOT/PSS-,and(D)PEDOT/PSS-PVA/PAA-depositedfilms.Insert:enlargedRamanspectrumbetween
1300and1600cm(cid:3)1of(B)PVA/PAA-,(C)PEDOT/PSS-,and(D)PEDOT/PSS-PVA/PAA-depositedfilms.
modified electrode emerged at (cid:3)0.35 V (vs. SCE). These results material, this implies that the PVA/PAA IPNs accelerated the
suggestthatamorehighlydopedstateoftheCP-hydrogelfilmswas deposition of CPs, which further enhanced its capacitance
formedwiththenearbycarboxylicacidgroupsascomparedtothe performance.
pureCPs,whichisinaccordancewiththeRamanresults.
After electrochemical deposition, the cathodic charge storage 3.2.3. Electrochemicalimpedancespectra
capacity (CSCC) increased from 1.5 mC/cm2 to 79 mC/cm2 and In order to record single neuron activity and reduce surgical
121 mC/cm2 for the PEDOT/PSS- and PEDOT/PSS-PVA/PAA-IPN- trauma,thegeometricsizeoftheneuralelectrodewasreducedto
modified microelectrodes, respectively. As the CSCC of the elec- themicro-scale,whichsharplyincreasedtheelectrodeimpedance
trode was in accordance with the total amount of electroactive and consequently decreased the qualityof signal recordings [21].
Fig.4. CyclicvoltammogramsofPtmicroelectrode((cid:5)(cid:5)(cid:5)(cid:5)(cid:5)(cid:5)),PEDOT/PSS-depositedPtmicroelectrode(┈┈)andPEDOT/PSS-PVA/PAA-depositedPtmicroelectrode(ddd).
Y.Luetal./Biomaterials33(2012)378e394 385
Impedance at 1 kHz is an important characteristic parameter for
neuralelectrodes,andisrelevanttoneuronalelectrophysiological
activity [43]. The electrochemical impedance spectra of the Pt
microelectrode and PEDOT/PSS- and PEDOT/PSS-PVA/PAA-IPN-
modified Pt microelectrodes were tested at their open-circuit
potentials in ACSF for assessing the electrochemical characteris-
ticsat theirinterfaces.AsshowninFig.5,theimpedancesof the
PEDOT/PSS- and PEDOT/PSS-PVA/PAA-IPN-modified microelec-
trodes at 1 kHz decreased from w51 kUe2.55 kU and 2.73 kU,
respectively, which is w95% lower than the unmodified Pt Fig.6. Proposedequivalentcircuitmodelforelectrochemicallydepositedelectrodes,
microelectrode. withcircuitelementsincludingsolutionresistance(RS),coatingcapacitance(CC),pore
resistance(RP),double-layerconstant-phaseelement(ZCPE),chargetransferresistance
An equivalent circuit model of the electrode-polymer- (RT),andfinite-lengthWarburghdiffusionimpedance(ZD).
electrolyteinterfaceisshowninFig.6,whichhasbeendiscussed
inpreviousworks[43].Thisequivalentcircuitmodeliscomprised rates,oranon-uniformcurrentdistribution.Whenn¼1,theCPEis
of circuit elements including the solution resistance (RS), coating identicaltoacapacitor.
capacitance(CC),poreresistance(RP),double-layerconstant-phase The finite-length Warburgh diffusion impedance (ZD) is intro-
element (ZCPE), charge-transfer resistance (RT), and finite-length duced in order to describe the ion diffusion within the polymer
Warburghdiffusionimpedance(ZD). films.ElementZDischaracterizedbythefollowingequation:
The constant phase element (CPE) represents the dissipative
double-layer capacitance, which reflects the characteristics of Z ¼ fR =ðjuC Þg1=2cothðjuR C Þ1=2; (5)
D D D D D
a microscopic fractal at the blocking liquidesolid interfaces. The
conceptoftheCPEisexplainedthroughthefollowingequation: where CD is the diffusional pseudocapacitance and RD is the
diffusionresistance.
(cid:1) (cid:3)
Z ¼ 1= qðjuÞn ; (4) The fitting data for the proposed equivalent circuit model are
CPE
giveninTable1.Asshowninthetable,theporeresistance(RP)ofthe
wherej¼O-1,uistheangularfrequency(rads(cid:3)1)¼2pf,andfisthe PEDOT/PSS-PVA/PAA-IPN-modified microelectrode is slightly
frequencyinHz.TheparametersoftheCPEaredefinedbyqandn. higherthanthePEDOT/PSS-modifiedelectrode.Thissuggeststhat
ParameterqindicatesthevalueofthecapacitanceoftheCPEasn thehydrogelcomponentoftheCP-hydrogelcompositepolymerfilm
approaches1,andhasanumericalvalueof1/ZCPEatu¼1rad/s.The mayhavereduceditsporosity,whichmayhinderthetransferofions
parameternrevealsthemicrofractalanddistributionofthepha- within the polymer film and subsequently increase the charge
seephase interface, which correlates toenergy dispersion on the transferresistance(RT).Thecoatingcapacitance(CC)increasedfrom
electrodeeelectrolyte interface and can be affected bya series of 0.14 mF for the PEDOT/PSS-modified microelectrode to 0.18 mF
factors,suchassurfaceroughness,thedistributionofthereaction for the PEDOT/PSS-PVA/PAA-IPN-modified microelectrode, which
Fig.5. ElectrochemicalimpedancespectraofPtmicroelectrode,PEDOT/PSS-depositedPtmicroelectrodeandPEDOT/PSS-PVA/PAA-depositedPtmicroelectrode.
386 Y.Luetal./Biomaterials33(2012)378e394
Table1 Furthermore, the CPE-n values of these deposited microelec-
FittingdataforproposedequivalentcircuitmodelshowninFig.7. trodesarecloseto1,suggestingthattheyarealsoclosetotheideal
Parameters Unit Pt PEDOT/PSS PEDOT/PSS-PVA/PAA capacitance.Thedouble-layercapacitance(CPE-p)anddiffusional
Z(at1kHz) kU 51.2 2.54 2.73 pseudocapacitance (CD) of the PEDOT/PSS-PVA/PAA-IPN-modified
RS kU 2.96 2.23 2.46 microelectrode are higher than for the PEDOT/PSS-modified
CC mF d 0.14 0.18 microelectrode.Thiscanprobablybeattributedtothecomponent
RP kU d 0.35 0.37 ofthePVA/PAAIPNhydrogel,whichworkednotonlyasascaffold
CPE-q mFs(cid:3)1 0.01 0.32 0.73 butalsoasadopant(eCOO(cid:3))forthecompositepolymerfilm.
CPE-n 0(cid:6)n(cid:6)1 0.85 0.93 0.88
RT kU d 0.15 0.23
CD mF d 0.048 0.068 3.2.4. Stabilitytest
RD kU d 13.7 14.6 Both electrochemical and mechanical stabilities are critically
importantforchronicallyimplantedneuralprobes,asthedamage
correlateswiththevariationinthecathodicchargestoragecapacity. fromtheelectrodesoradelaminationofthecoatingmaterialmay
ItalsoimpliesthatthedepositedPEDOT/PSS-PVA/PAAIPNfilmis harm the surrounding tissue and sharply bring down the perfor-
thickerthanapureCPfilm,thusresultinginanincreaseddiffusion manceoftheimplantedmedicaldevice.Inordertoinvestigatethe
resistance(RD). electrochemical and mechanical stabilities, the PEDOT/PSS- and
Fig.7. Cyclicvoltammogramsof(A)PEDOT/PSS-and(B)PEDOT/PSS-PVA/PAA-depositedmicroelectrodesbefore(ddd)andafter(┈┈)stabilitytest.
Y.Luetal./Biomaterials33(2012)378e394 387
Fig.8. SEMimagesofdepositedPVA/PAA(A,D),PEDOT/PSS(B,E),andPEDOT/PSS-PVA/PAA(C,F)filmsonelectrodesunderoptimizedconditions.In(A,B,C)thescalebaris1mm;
in(D,E,F)thescalebaris400nm.
PEDOT/PSS-PVA/PAA IPN-modified microelectrodes were cyclic CSCClossincreasedto95%(showninFig.7(A)).Onepossiblereason
voltammetricscannedbetweenthepotentialsof(cid:3)0.6Vand0.6V for this degradation of the deposited film is the volume change
(vs. SCE) at a scan rate of 50 mV/s for 100 cycles, followed by causedbythemasstransportduringcyclicvoltammetricscanning
immersioninanACSFfor1week. [44]. The volume change may subsequently result in mechanical
Aftercyclicvoltammetricscanning,theCSCClosswas15%and8% instabilityaftertheelectrochemicalstabilitytests,suchasdelam-
for the PEDOT/PSS- and PEDOT/PSS-PVA/PAA-IPN-modified micro- ination or poor adhesion of the deposited films to the electrode
electrodes,respectively.ThelossofCSCCmaybeduetotheirreversible substrate. However, in contrast with PEDOT/PSS films, the cyclic
reactionsofthePEDOTbackbone,whichrepresentsadeterioration voltammograms of thePEDOT/PSS-PVA/PAA-IPN-modified micro-
of the electrochemical reversibility [29]. This suggests that the electroderemainedalmostunchangedafterthestabilitytests,and
PEDOT/PSS-PVA/PAA-IPN-modifiedmicroelectrodedemonstratedan thetotalCSCClossremainedat8%(showninFig.7(B)).
improvedelectrochemicalstabilityascomparedtothepureCPs. Althoughtheelectrodepositionforeachtypeoffilmwascarried
AfterimmersionintheACSFfor1week,seriousdelaminations outunderoptimizedconditions,itisobviousthatthePEDOT/PSS-
wereobservedonthePEDOT/PSS-modifiedmicroelectrode,andthe PVA/PAA-IPN-modified microelectrode exhibits much improved
Fig.9. PC12cellsculturedonITOsubstrate(A,D),PVA/PAA-coatedITOsubstrate(B,E),andPEDOT/PSS-PVA/PAA-depositedITOsubstrate(C,F)inpresenceof50mg/mLofNGFon
seventhday.Thescalebaris50mm.