Biochimica et Biophysica Acta 1830 (2013) 4334–4344

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Biochimica et Biophysica Acta

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Review

Organic bioelectronics for electronic-to-chemical translation in modulation ofneuronal signaling and machine-to-brain interfacing☆

Karin C. Larsson, Peter Kjäll, Agneta Richter-Dahlfors ⁎Swedish Medical Nanoscience Center, Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden

☆ This article is part of a Special Issue entitledOrganic Bioin Biomedicine.⁎ Corresponding author at: Department of Neuroscienc

77 Stockholm, Sweden. Tel.: +46 8 524 874 25; fax: +4E-mail address: Agneta.Richter.Dahlfors@ki.se (A. Ri

0304-4165/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbagen.2012.11.024

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 10 September 2012Received in revised form 14 November 2012Accepted 27 November 2012Available online 6 December 2012

Keywords:Organic bioelectronicsDrug deliveryCa2+ signalingSpatial-temporal gradientIn vivo

Background: A major challenge when creating interfaces for the nervous system is to translate between thesignal carriers of the nervous system (ions and neurotransmitters) and those of conventional electronics(electrons).Scope of review: Organic conjugated polymers represent a unique class of materials that utilizes both electrons andions as charge carriers. Based on thesematerials,we have established a series of novel communication interfaces be-tween electronic components and biological systems. The organic electronic ion pump (OEIP) presented in this re-view is made of the polymer–polyelectrolyte system poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)(PEDOT:PSS). The OEIP translates electronic signals into electrophoretic migration of ions and neurotransmitters.Major conclusions: We demonstrate how spatio-temporally controlled delivery of ions and neurotransmitters canbe used to modulate intracellular Ca2+ signaling in neuronal cells in the absence of convective disturbances. Theelectronic control of delivery enables strict control of dynamic parameters, such as amplitude and frequency of

Ca2+ responses, and can be used to generate temporal patterns mimicking naturally occurring Ca2+ oscillations.To enable further control of the ionic signals we developed the electrophoretic chemical transistor, an analog ofthe traditional transistor used to amplify and/or switch electronic signals. Finally, we demonstrate the use of theOEIP in a new “machine-to-brain” interface by modulating brainstem responses in vivo.General significance: This review highlights the potential of communication interfaces based on conjugated poly-mers in generating complex, high-resolution, signal patterns to control cell physiology. We foresee widespreadapplications for these devices in biomedical research and in future medical devices within multiple therapeuticareas. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The brain is the central cognitive unit bywhich sensory input aswellas all other internal and external information is processed. In roughanalogy, the brain can be compared to a computer, as both are informa-tion processors identical in their use of mobile charge carriers andelectric signals to convey information. Neurons, the basic informationprocessing units of the brain, send electrical signals (action potentials)and utilize ions and ion fluxes as major charge carriers. Computers onthe other hand, transfer information by moving charge in the form ofelectrons. Although the mobility of electrons in silicon is 106 timesfaster as compared to ion mobility in water, the transport of chargerepresents a crucial link between these biological and electronic sys-tems. In this way, the concept of electronic and ionic signal transport

electronics—Novel Applications

e, Karolinska Institutet, SE-1716 8 34 26 51.chter-Dahlfors).

l rights reserved.

will be central in establishing new communication interfaces bridgingthe gap between electronics and biological systems.

Organic conjugated polymers represent a class of materials thatuniquely utilize both electrons and ions as charge carriers. In this re-view, we present the basics of this relatively new transport technology,and how it can be implemented as novel communication interfacesbridging electronic components and neurobiology. As the basics ofneuronal signaling form the foundation for device development, thefirst sections of this review give a brief description of the most relevantconcepts of neurobiology. Next, the reader is guided through a briefsummary of contemporary techniques used to deliver stimuli in cellsignaling studies, before the function of organic bioelectronics devicesand their use in neurobiology is described in detail.

2. Neuronal cell signaling

Cell signaling is part of a complex communication system that gov-erns basic cellular activities. From the moment of fertilization through-out life, networks of intra- and extracellular signaling pathways conveyinformation delivered by signal carriers such as biomolecules and ions.Cellular signal transduction consists of a number of steps, beginning

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with the arrival of a signal that activates the cell. The activation mayoccur by a molecule binding to a receptor on the cell surface, ionschanging the membrane potential of the cell or a shear flow mechani-cally activating the cell. The signal is conveyed to trigger intracellularpathways of ions and molecules, propagating the signal inside the cellto its intended destination. The signal finally arrives at its downstreamtarget and changes the state of a cell, for example by altering geneexpression patterns by regulation of transcription factors.

The nervous system consists of billions of neurons and an equallygreat number of supporting cells. Neurons are highly specialized inprocessing and transmitting cellular signals and they are organized inneuronal circuits controlling and coordinating functions of sensation,perception and behavior. In other words, a vast number of events inthe human body rely on neurons communicating with one another.Neurons are excitable cells and their communication is dependent onboth chemical and electrical signal transmission (Fig. 1). The translationbetween chemical and electrical signals is dependent on a number ofdifferent ions and chemical messengers, so called neurotransmitters.In the resting state, ion selective pumps in the neuron's cell membranebuild up an ionic concentration difference, making the inside of the cellmore negatively charged compared to the outside. This results in anelectrochemical potential difference over the membrane, which isknown as the resting potential. When a neuron is stimulated, openingof ion channels result in ion fluxes across the cell membrane changingthe membrane potential. If the unbalance of charge causes the mem-brane potential to exceed a certain threshold, the neuron is activatedand fires an action potential. As the flux of ionic charges moving acrossthe membrane, the action potential can be considered an electricalsignal conducted along the axon. When the action potential reachesthe axon terminals, Ca2+ enters the cell and triggers exocytosis. Duringthis process, synaptic vesicles on the transmitting presynaptic neuronfuse with the cell membrane and release neurotransmitters into theextracellular space of the synaptic cleft. The neurotransmitters diffuseacross the 20 nm wide gap of the synapse and activate receptors onthe receiving postsynaptic cell. If the target cell is another neuron theprocess can start all over again.

Acetylcholine (ACh) is one of the major neurotransmitters in thenervous systems. Neurons containing ACh, so called cholinergic cells,are involved in signaling in both the central and peripheral nervoussystem (CNS and PNS). In the CNS, cholinergic neurons are located inthe basal fore brain fromwhere they project to the hippocampus, amyg-dala and cerebral cortex [1]. In the PNS, ACh plays an important role inthe synaptic transmission at the skeletal neuromuscular junctions [2].The cholinergic receptors are defined as nicotinic acetylcholine recep-tors (nAChRs) and muscarinic acetylcholine receptors (mAChRs). ThenAChR, named for its affinity to the natural occurring CNS stimulantnicotine obtained from the tobacco plant, is an ionotropic nonselectivetransmembrane cation channel. The metabotropic mAChR, a G-protein

Fig. 1. Neuronal cell signal transmission. The neuron consists of the soma, the cell body, denthat transmits the signal from the cell body to the target cell. Information from the chemicaand is converted back to chemical output at the axon terminals.

linked receptor, is named for its activation bymuscarine, which is a poi-sonous chemical naturally found in mushrooms. Changes in cholinergicsignaling or even loss of cholinergic neurons are observed in a numberof progressive neurodegenerative disorders such as Alzheimer's diseaseand Parkinson's disease [3,4]. Development of therapies has thereforebeen targeted towards themolecular players involved, especially inhib-itors of ACh esterase or agonists to nAChRs and mAChRs [5,6]. Under-standing of the molecular mechanisms and functions of cholinergicsignaling has greatly benefitted from the use of endogenous AChas well as cholinergic agonists such as natural chemical compounds,e.g. nicotine and muscarine.

2.1. Calcium signaling

Ion fluxes are of crucial importance in all living systems. The Ca2+

ion plays a vital role as a highly versatile second messenger that regu-lates many different cellular responses and functions. Events such asmuscle contractions, cell migration and fertilization are all known todepend on Ca2+ signaling and the accompanied signal transductionpathways [7]. Ca2+ signals are often organized in complex spatial andtemporal patterns in order to create signals with different durationand concentration ranges. These signals are commonly referred to asoscillations, waves, spikes, or puffs. The periodic behavior of the Ca2+

signal is essential for cell survival since an elevated, sustained concen-tration of intracellular Ca2+ is toxic for the cell [8]. In the synaptic junc-tion, Ca2+ oscillation occurswith a periodicity in themicrosecond rangeto trigger exocytosis, whereas events such as gene transcription and cellproliferation involves Ca2+ signaling operating in the minutes to hoursrange [7].

The Ca2+ signaling systems of mammalian cells utilize an extensivesignaling toolkit to both receive and present highly complex signalingpatterns. This advanced machinery consists of sensory mechanisms,channels and transporters that together regulate the gradients acrossthe plasmamembrane and intracellular stores (Fig. 2). In the eukaryoticcell the resting intracellular free Ca2+ concentration ([Ca2+]i) is main-tained at approximately 100 nMwhile the extracellular [Ca2+] exceeds1 mM. Cells also contain intracellular stores of free Ca2+ (in the mMrange) that primarily are located in the sarcoplasmic reticulum (SR)or endoplasmic reticulum (ER). When information has to be retainedover longer periods of time, the Ca2+ signaling system use repetitivetransients, i.e. [Ca2+]i elevations followed by rapid decays [7]. Ca2+

oscillations have been described in analogy with the amplitude modu-lation (AM) and frequency modulation (FM) in electronic communica-tion, where AM refers to differences in [Ca2+]i signal strength whereasFM refers to the interval of [Ca2+]i elevations [9]. These integratedpatterns create specificity for diverse cellular mechanisms and activa-tion of a range of biological processes including cytokine release in

drites, processes from the cell body receiving synaptic input, and an axon, the processl input is translated into an electrical impulse, relaying the message along the neuron,

Fig. 2. Cellular regulators of Ca2+ signaling. Channels regulating Ca2+ entry are either voltage operated Ca2+ channels (VOCCs) or receptor operated channels (ROCCs). When the[Ca2+]i increases pumps, exchangers and buffers will remove Ca2+ from the cytoplasm in order to avoid toxicity. Plasma-membrane Ca2+-ATPases (PMCAs) use ATP to pump Ca2+

against the electrochemical gradient and in Na+/Ca2+ ion exchangers (NCXs) one cytoplasmic Ca2+ ion is exchanged for three extracellular Na+ ions. Release of Ca2+ from intracellularstores occurs from the inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). Re-filling intracellular stores with Ca2+ is performed by channels located on the SR/ER known as SR Ca2+-ATPases (SERCAs). Re-filling of the stores is also accomplished by an intracellular event, triggering opening of store operated Ca2+ channels (SOCC) in the plasmamembrane.

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renal proximal tubule cells, oocyte activation and determination of cellneurotransmitter phenotype [10–13].

2.2. Contemporary techniques to stimulate cells in vitro and in vivo

To elucidate the mechanisms involved in specific cell signalingevents, cell biologists expose cultured cells and tissues to defined stim-uli. Alternatively, experiments are performed in a variety of animalmodels. Neuronal cells can be stimulated by a number of chemical sub-stances (e.g. neurotransmitters and drugs) as well as factors of physicalorigin, such as pressure, temperature, light and electric fields. By induc-ing signaling pathways with appropriate stimuli it is possible to charac-terize the players participating in the dynamic cell signaling processes.These players provide clues that aid in understanding of cellular physi-ology and possibly in the development of treatment for neurophysio-logical disorders. Among the most commonly used techniques toinvestigate cellular signal transduction pathways in vitro are live cellimaging and electrophysiological approaches. The different types oftechniques used to stimulate single or multiple cells in these set-upscan be defined as flow based and non-flow based (Table 1). Some ofthese methods are also valuable research tools for in vivo experiments;

Table 1Examples of delivery techniques used for stimulation of cellsa.

Technique Possibility to calculatedelivered concentration

Spatial resolution

Flow basedBath application ++b –

Puffer pipette ++ ++Microfluidics ++ +

Non-flow basedElectric stimulation − –

Uncaging + +++Optogenetics − +++Iontophoresis + ++

a Table compiled partly based on information from references.b n/a=not applicable; -=low; +=good; ++=very good; +++=excellent.

however, only a few techniques currently have the potential to bedeveloped or integrated into new medical devices.

2.2.1. Flow-based delivery of stimuliIn themajority of contemporary delivery techniques, the stimuli are

dissolved in a liquid, which is introduced to the target system by liquidflow. Most commonly the liquid is manually added by a pipette or viaflow chambers and perfusion systems controlled by motor- or gravity-driven pumps. However, any kind of liquid flow will inevitably exposecells to shear stress. Also, as cell-secreted factors important in cell-to-cell communication may be washed away, flow-based deliveryhampers from the fact that it may alter the cellular microenvironment,potentially changing the state of the cell [14]. In bath applications, theentire cell population is stimulated, and to achieve higher spatial reso-lution the delivery has to be refined. Local delivery of stimuli can beachieved by pressure-ejection from a fluid filled glass pipette, alsoknown as a puffer pipette [15]. As the delivered solution mix with thebath solution it is difficult to determine the exact concentration arrivingat the cell and leakage from the glass micropipette tip can result inproblems when recording baselines. The puffer pipette technique is es-pecially valuablewhen stimulating only one or a few cells in an explant,

Temporal resolution Clinical applicability References

+ n/a [45,46]++ n/a [15,26,47]+ + [14,48,49]

++ +++ [16,17,50]+++ n/a [20,21,51]+++ n/a [18,19]+ ++ [22,23,25]

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e.g. brain slice, as the local delivery allows for several experiments in thesamepreparation and low consumption of potentially expensive stimu-li. The development of small, portablemicrofluidic systems has enabledfurther reduction of volumes and consumption of delivered stimuliin various cell signaling studies. However, operation of microfluidicdevices requires connections to tubes, valves and external pumps,which makes this approach altogether bulky and in some perspectivesunfitting for development towards implantation.

2.2.2. Non-flow based delivery of stimuliExcitable cells respond to depolarizing electric fields and some of

today's most well-established neural prostheses are based on electri-cal stimulation of cells and tissues. The cochlear implant bypasses thedamaged part of the ear and provides direct electronic stimulation ofthe auditory nerves. In deep brain stimulation, surgically implantedprobes send electrical impulses to suppress symptoms in patientswith for example Parkinson's disease [16]. Electrical stimulation isbased on depolarization of the semi-permeable cell membranes at theaxon of neuronal cells and subsequent generation of action potentials[17]. As electric stimulation bypass the chemical input it does notdiscriminate different types of cells in the vicinity of the electrode. Toobtain a more specific physical activation, photostimulation techniquesbased on light-activation of chemically or genetically modified recep-tors or ion channels can be used (optogenetics) [18,19]. This type ofoptical stimulation can be used for cell-type-specific stimulation toinduce hyperpolarizing or depolarizing cell responses with high spatio-temporal resolution. A second type of photostimulation techniqueresults in chemical stimulation by light-mediated release, or uncaging,of biologically inactivated compounds. The caged compounds, whichcan be ions, neurotransmitters or other signalingmolecules, are floodedover the preparation [20]. When illuminated with light of definedwavelength, the caged groups absorb photons, breaking the bondbetween the caged group and the stimuli, causing release of the stimuliin the intra- or extracellular environment. Combining cages containingdifferent compounds that are activated at individual wavelengths, gen-eration of highly complex multi-site activation patterns can be generat-ed [21]. Although the photoactivation methods will remain valuableresearch tools both in vitro and in vivo, their inherent limitations pre-vent clinical staging. Yet another non-flow based delivery technique isiontophoresis, currently in clinical use as a non-invasive transdermal de-livery of charged molecules for localized treatments and systemic targets[22,23]. Iontophoresis is best described as a type of electrophoresis, wherea charged field is created between two electrodes in a solution and smallcharged ions or molecules are delivered by electromigration and electro-osmosis (current-induced convective flow of water) [24]. The same prin-ciple is applied when the technique is used for fine-tuned delivery ofchemical substances in vitro. These types of micro-iontophoresis systemscan be a glass micropipette or a container on a carbon fiber microelec-trode [25,26]. Applying a potential over the ion solution, a constant elec-tric field will cause ions to move out from the tip of the micropipette.

Collectively, the techniques presented here represent examples ofmethods used in cell signaling studies in vitro and in vivo. Flow-basedtechniques provide good control over applied concentrations butinduce convection in the target system and generally have less spatio-temporal resolution compared to the non-flow based methods. On theother hand, stimulating cells with highly spatiotemporal non-flowmethods is often complicated and it is difficult to quantify amount ofapplied stimuli. Hence, there is a need for a non-flow based deliverytechnology by which a defined number of molecules can be deliveredwith high spatio-temporal resolution.

3. Organic bioelectronics as delivery devices for bio-substances

Bioelectronics is an interdisciplinary research field where ele-ments of physics, electronics, materials science and biology merge. Or-ganic bioelectronics is a carbon-based (therefore defined as organic)

technology. The active part of an organic bioelectronics device is usuallycomprised of conjugated polymers, alone or in combination withothermaterials to form a primary or secondary interfacewith biologicalspecimens or bio substances (therefore defined as bio-). The conductiv-ity of the polymermaterialmakes it possible to design devices that havea similar functionality as classical electronics (therefore defined aselectronics). A key feature of most conducting polymers is the propertyof both electronic and ionic conductivity, which makes them a naturalcandidate to translate between the electron-based world of classicalelectronics and the generally ion- and molecular-based world ofbiology. The applications of organic bioelectronics are diverse; henceits many features that can, alone or in combination, provide distinctlydifferent advantages to biomedical methodologies. The field of“Organic Bioelectronics” was defined in 2007 [27] and the focus ofthis emerging field is the development of novel communicationinterfaces for monitoring and regulating cellular functions.

3.1. The organic electronic ion pump (OEIP)

Taking advantage of the before mentioned properties of conjugat-ed polymers a new type of bioelectronic device was developed in2007—the organic electronic ion pump (OEIP) [28]. The OEIP isbased on poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate)(PEDOT:PSS), a chemically stable polymer–polyelectrolyte systemwith combined electronic and ionic conductivity (Fig. 3). While theconductive properties ofmetals and inorganic electronics depend solelyon electrons, conjugated polymers such as PEDOT:PSS can conduct bothelectrons and ions. PEDOT provides electronic conductivity, while PSSprovides both enhanced electronic conductivity and cationic conductiv-ity. In the OEIP device, a PEDOT:PSS film is patterned onto a polyethyl-ene terephthalate (PET) substrate (Fig. 4). The pattern consists oftwo electrodes, a source and a target, connected by a polymer channel.Over-oxidation of the channel disrupts the conjugation pathway of thePEDOT backbone, rendering the channel electronically insulating. As theover-oxidation process leaves the PSS phase intact, this creates a chan-nel that conducts ions but not electrons. The channel and electrodes arecovered by a hydrophobic photoresist, which provides openings forapplication of electrolytes on the two electrodes as well as for contactpoints for control electronics. The source electrolyte contains the posi-tively charged ion to be delivered into the target electrolyte. Whenaddressing the OEIP, redox reactions dependent on transport of bothmobile cations (M+) and electronswill take place. The source electrodeis oxidized and the target electrode is reduced according to the electro-chemical half-reactions:

PEDOTo þMþPSS−→PEDOTþPSS− þMþþ e− ðReaction 1aÞ

PEDOTþPSS− þMþ þ e−→PEDOTo þMþ þ PSS− ðReaction 1bÞ

Oxidation of the source electrode (Reaction 1a) will result in M+

from the source electrolyte to enter the source electrode (anode). Thefall of potential over the ionically (but not electronically) conductivechannel will result inmigration of M+ towards the reduced target elec-trode (cathode, Reaction 1b). As M+ reach the rim of the hydrophobicresist by the channel outlet, M+ will be released into the target electro-lyte and spread by diffusion. Since the delivery of M+ does not rely onaqueousflow, no convective disturbances are induced in the target elec-trolyte. Given by the electrochemical relationships of the equations,each transported ionic charge M+ is compensated by an equal amountof transported electronic charge transferred between the electrodes.Hence, the currentmeasured in the electronic branch of the circuit is di-rectly proportional to the delivery rate of cations in the target electro-lyte. This novel technique enables a unique way to electronicallycontrol the lateral transport and delivery of positively charged ions toan electrolyte.

A

B

Fig. 3. Chemical structures of (A) neutral poly(3,4-ethylenedioxythiophene) (PEDOT) and (B) PEDOT doped with poly(styrenesulfonate) (PSS) making up the polymer–polyelectrolyteblend PEDOT:PSS.

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3.2. OEIP transport of ions and biomolecules

The first OEIP, published in 2007, demonstrated electronically con-trolled transport and delivery of the smallest positive ion, the proton

Fig. 4. Schematic side view of the OEIP. The black arrow represents the transport ofcharged ions M+ upon electrical addressing of the device. M+ migrates from the sourceelectrolyte, into the anode, through the over-oxidized channel, and out into the targetelectrolyte on top of the cathode. The electrochemical potential fall is confined to theover-oxidized channel. Figure is reproduced from Ref. [30].

H+, and the metal ions K+ and Ca2+ [28,29]. The successful transportof small metal ions inspired us to investigate if other biomoleculesrelevant for neuronal cell signaling could be transported through theOEIP. In Table 2, these substances are summarized, along with theirselected function, chemical structure and transport efficiency (for de-tailed description, see below).

When PEDOT:PSS undergoes a redox reaction (Reaction 1a,b), theelectronic charge transferred in the reduction of the polymer isbalanced by an equal amount of ionic charge transported throughthe over-oxidized polymer-channel. The mobility of each transportedion in the channel is reflected by its net-charge, physical size and chem-ical structure. Together these parameters determine the transportefficiency of the ion and accordingly how effectively it will be delivered.The transport efficiency can be defined as the molecule-to-electronratio, in which the total number of ions transported through thechannel is compared to the total number of electrons transferredthrough the electronic branch of the circuit. The total number of elec-trons is calculated from the total charge transported in the drivingcircuit, which is obtained from recorded currents. To determine thetotal number of molecules delivered through the channel, differentmethods can be applied, such as high-performance liquid chromatogra-phy (HPLC) and fluorometric enzyme assays. Transport efficiencies forthe signaling species known to be transported in the OEIP are presentedin Table 2.

In an optimal situation, each electron measured in the electronicpart of the circuit corresponds to the delivery of one ion, giving amolecule-to-electron ratio of 1 and a transport efficiency of 100%. Thisnumber is, however, shown to varywidely depending on the substance.The low transport efficiencies of the neurotransmitters glutamate (Glu)

Table 2Delivery repertoire and efficiencies of the OEIP.

Signaling species Selected functions Chemical structure Transportefficiency

Ref.

IonsNa+ Involved in the

initiation of actionpotentials.

80% [34,52]

K+ Maintain theresting potential inexcitable cells.

100% [28]

Ca2+ Important secondmessenger in cellsignaling, dictatesrelease ofneurotransmitters.

100% [28]

NeurotransmittersAcetylcholine Neurotransmitter

in the CNS and PNS.100% [34]

Aspartate Excitatoryneurotransmitterin the CNS.

16% [30]

GABA Major inhibitoryneurotransmitterin CNS.

77% [30]

Glutamate Major excitatoryneurotransmitterin the CNS.

37% [30]

A

B

Fig. 5. (A) Intracellular Ca2+ recordings of HCN-2 neuronal cells depolarized with OEIPtransported K+ (dashed line), in the presence of general Ca2+-channel inhibitor GdCl3(solid line) or VOCC inhibitor, nifedipine (dotted line). (B) Visualization of the pH gradientin the target electrolyte after OEIP transport of H+, deep red color indicates pH ~2 andclear yellow pH ~5. Figure A is reproduced from Ref. [28].

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and aspartate (Asp) can be explained by the fact that these two aminoacids are transported in an excess of H+ in the source solution [30]. Dis-solving Glu/Asp in water will result in an acidic solution and Glu/Aspmolecules will exist in both protonated and de-protonated forms. TheGlu/Asp molecules with a net positive charge will have to competewith the small H+ ions, which have higher mobility in the channeland therefore will be more readily transported [29].

OEIP operation can be performed in constant voltage mode, with aDC voltage applied between the two electrodes, or in a constant currentmode, when a DC current is applied to the device. When the OEIP isactivated by electronic addressing, recordings of potentials/currentsduring operation of devices demonstrate stable delivery over time.Furthermore, this demonstrates that activation of the device doesnot result in burst release, a phenomenon commonly encountered indifferent types of nanostructured delivery systems. In drug-loaded con-jugated polymer actuator systems, which contract when electricallyaddressed, an initial rapid release is often followed by a sustainedslower delivery profile [31,32].

One important factor to considerwhen deciding on delivery systemsfor a specific application is whether leakage of stimuli occur when thesystem is switched to its off-mode. When the OEIP is turned off, i.e. nopotential or current is applied, the diffusion-mediated leakage fromthe source electrolyte to the target electrolyte is very small. The leakageof ACh was found to be below the detection level of used measuringmethod (0.3 μM) and previously K+ delivery showed an on/off ratioin delivery rate higher than 300 [28]. Hence, turning the OEIP to theoff-mode, leakage of stimuli is negligible. This distinct off-modeprovides a clear advantage of molecular and ionic delivery comparedto the push-and-pull methods of other techniques e.g. in iontophoresis,where leakage of stimuli from themicropipette is usually circumventedby applying a retaining current, i.e. reversing the current [26], ormicrofluidics systems, where leakage problems are often solved byreciprocal pumping systems [33]. Compared to other delivery tech-niques the OEIP can also easily be switched back on for another roundof delivery.

3.3. Cell stimulation in vitro

Since OEIP signal translation mimic signal transmission in excitablecells (electronic input signals are converted to chemical output signals)(Fig. 1), theOEIPwasused to precisely control signaling pathways in ex-citable cells. The first bio-relevant ion demonstrated to be transportedthrough the OEIP was the monovalent metal ion K+, which amongother cell signaling mechanism is involved in dictating the membranepotential in neuronal cells [28]. Using the planar OEIP depicted inFig. 4, a solution containing K+ was placed on top of the source elec-trodewhereas adherent neuronal cells were cultured on the target elec-trode in cell media. Applying a potential between the source and targetelectrodes initiated delivery of K+ through the 4 mm wide polymer-channel. After being released at the channel outlet, K+ diffuses intothe medium, causing a high local concentration (20–50 mM), whichdepolarizes the cell membrane and activates the membrane-boundvoltage-operated Ca2+ channels (VOCCs). The resulting cellular influxof Ca2+ was monitored using microscopy-based real-time Ca2+

imaging of cells pre-loaded with the Ca2+-sensitive probe Fura-2 AM(Fig. 5A). By applying VOCC-specific inhibitors to the cells, the Ca2+

response was abrogated. This demonstrates that the observed Ca2+

response represents a physiologically relevant cellular response todepolarization caused by the OEIP-delivered K+.

As delivery of ions is spatially confined to the point of the OEIP chan-nel outlet, ions diffusing from this point into the electrolyte will estab-lish a concentration gradient in the target electrolyte. This was shownin experiments where H+ was transported to the target electrolyte,

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where a standard pH indicator paper was placed on top of the targetelectrode [29]. Seconds after the onset of delivery, a gradient of H+,resulting in the decrease of 3 pH units, was observed (Fig. 5B). Electron-ic control of the on/off of the device also demonstrated that dynamicH+

oscillations can be achieved [29]. This indicated the potential of the de-vice in mimicking the oscillatory signaling patterns present in neuronalcells.

3.4. Modulation of cell signaling

Amajor neurotransmitter of the nervous system is ACh, a quaterna-ry ammonium compound, whichmaintains a positive charge across thefull pH range. The combined structural and functional properties of thisneurotransmitter made ACh an interesting candidate to test for OEIPtransport. Using the planar device depicted in Fig. 4, successful AChtransport was demonstrated. However, the high transport efficiency ofthis compound generated very high local [ACh], far higher than thephysiological relevant range. To reduce the number of transportedACh molecules, the polymer channel had to be miniaturized. Thus, adevice was designed with a 10 μm wide delivery channel (Fig. 6A).Additionally, a waste-electrode was introduced to improve on the tem-poral control of delivery. At the start of experiment, the channel waspre-filled by applying a potential between the source and the waste

A

B

Fig. 6. (A) Design of the 10 μm-OEIP. The source (S), waste (W) and target (T) electrodesof electronically conducting PEDOT:PSS are connected by the cation selective channel(pink). ACh (red) is delivered at the 10 μm channel outlet where it spreads by diffusionto the SH-SY5Y cells (green) cultured on top of the target electrode. (B) Temporal dynam-ics of Ca2+ signaling in cells located at 50 μm and 150 μm from the 10-μm channel outleton delivery of ACh. Voltage pulses of 20 V for indicated times generated Ca2+ oscillations.Reproduced with permission from Ref. [34], courtesy of Wiley-VCH Verlag.

electrodes (VS-W=20 V) for 10 min. Delivery of ACh to the cells wasthen initiated by applying a potential between the source and targetelectrodes. The pre-filling was found to dramatically improve on thetemporal control of the device (from minutes to seconds), as this steppositioned molecules at a point much closer to the target outlet wherecells are located. Pre-filling of the system also decreased delivery ofany undesired residual ions from the polymer material in the channel.As an added value, narrowing the channel to 10 μm also improved onthe spatial resolution for delivery, since this dimension allowed forsingle cell addressing.

Utilizing conjugated polymer devices as communication interfacebetween electronic components and the nervous system requires thatthe transported compound retain its biological activity after beingtransported through the polymer. To analyze the biologic activity oftransported ACh, human SH-SY5Y neuroblastoma cells, known toexpress the AChRs,were used as biosensors. By recording the intracellu-lar Ca2+ response in cells located next to the outlet point of the 10 μmdelivery channel, it was confirmed that ACh retain its biological activityafter transport [34].

When the potential is turned off, AChdelivery stops. Due to diffusionof ACh into the electrolyte, the high local concentration at the deliverypoint is rapidly decreasing. In addition to providing an opportunity toestablish local, controlled molecular gradients in the target electrolyte,controlled delivery versus diffusion also allows for the generation ofan AM/FM output signal. The AM signaling pattern in cells can thus becontrolled by increasing the applied voltage for a given pulse length,resulting in an increased delivery rate of ACh. Given that cells are stim-ulated with physiological concentrations of ACh that do not causedesensitization, an increased amplitude of the cellular Ca2+ responsecan be observed for each extended pulse length [34]. Correspondingly,prolonging the pulse length at a constant voltage will increase the am-plitude of the Ca2+ response, i.e.modulation of the FM signaling patternof the cell. Fig. 6B shows that along with an increase in pulse lengths(from 0.2 s to 2 s), the amplitude of the Ca2+ responses elicited in thecell located 50 μm from the channel outlet is enhanced. Moreover, thesame experiment shows that whereas a Ca2+ response is triggered inthe cell located closest to the channel outlet by ACh delivered by apply-ing a 20 V pulse for 0.2 s, the cell located 150 μm from the outlet onlyresponded when the pulse length was extended to 2 s. This demon-strates that the 10 μm OEIP delivery device can be used to modulationof ACh-delivery to single cells with a spatial resolution of 100 μm(Fig. 6A, insert).

Similar to the above, the 10 μmOEIP can be used to generate tempo-ral patterns that mimic naturally occurring Ca2+ oscillations in cells. Bymatching the length and strength of the applied pulse of ACh-deliverywith the Ca2+ response, repetitive Ca2+ spikes were elicited in an indi-vidual cell with a periodicity of about 100 s (Fig. 6B). This demonstratesthat the current version of the OEIP technique can be useful in applica-tions where modulation of the FM component of Ca2+ responses in thesecond to minute range. Examples of biological processes regulated byCa2+ fluctuations in this range include oocyte activation at fertilization,determination of cell neurotransmitter phenotype and neuronal cellmigration [11,12,35,36].

In summary, electronic control of molecular transport using theOEIP technology can hypothetically be used to fine-tune the deliveryto release of single molecules. As this yield a strict control of cellularsignaling patterns, it can be foreseen that the OEIP technology willset the stage for experimental designs based on complex addressingschemes.

3.5. A chemical transistor fine-tunes ion fluxes

To enable the construction ofmore advanced delivery schemes, newmethods of controlling ion flows are required. In conventional electron-ics, transistors are active circuit elements used to amplify and/or switchelectronic signals. In analogy to the electronic transistor, we developed

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an electrophoretic chemical transistor, the “ion bipolar junction transis-tor” (IBJT) [37]. The IBJT consists of three channels (emitter, collectorand base), which are connected to terminals of PEDOT:PSS (Fig. 7A).The cation selective emitter and collector channels consist of over-oxidized PEDOT:PSS, while the base channel is made of an anion selec-tive membrane. The three channels meet in a junction where they allinterface a neutral cross-linked gel layer. Transport of ACh from theemitter to the collector requires that voltage is applied across theemitter-collector (VEC>0 V) and that the junction is conductive. Thelatter is obtained by varying the salt concentration in the cross-linkedgel layer. In the active mode (VEB>0 V), chloride ions (Cl−) migratethrough the base into the junction where they get compensated bycations (ACh) from the emitter. This increases the ionic conductivitybetween the emitter and collector, allowing positively charged AChmolecules to be transported from the emitter, through the gel and tothe collector. Hence, the ionic current between the emitter and collec-tor, i.e. the amount of deliveredACh, becomes a function of the potentialapplied to the base.

The functionality of the IBJT was demonstrated using real-timeCa2+ imaging recordings of adherent SH-SY5Y cells cultured by theoutlet on the collector electrode. Baselines of [Ca2+]i were recordedwith the IBJT in its off state. By reversing the biased voltage of thebase, the device is switched to its active mode. As Cl− migratethrough the base channel into the junction, migration of ACh fromemitter to collector is achieved. By monitoring the cellular Ca2+ re-sponse, delivery of ACh to cells located at the outlet on the collectorelectrode was demonstrated (Fig. 7B). Turning the IBJT off, Cl− migratefrom the junction, back to the base. This terminates ACh-delivery andfluorescence from the Ca2+-indicator decrease to a lower level. Thisexperiment demonstrates how an anionic (Cl−) base-current can beused to turn ACh-delivery on and off in the purpose of controllingACh-delivery to modulate Ca2+ signaling. The delay in the Ca2+

response after turning the base on can be explained by the timerequired for building up the concentration of Cl− in the junction. Corre-spondingly, the slow decay in fluorescence is a result of the timerequired to stop ACh-transport by depleting the junction from Cl−.This experiment demonstrates how the inherent biological specificityof the ionic charge carrier (ACh) enables targeted on/off cell stimulationbased on elementary transistor principles.

The pnp-IBJT was recently followed up by the development of theanionic equivalent, the npn-IBJT, controlling delivery of negativelycharged ions [38]. Further, by connecting several IBJTs in a circuit, in-tegrated chemical logic gates have been demonstrated [39]. The two

Fig. 7. The pnp-IBJT. (A) The emitter, collector and base channels meet in a junction consistingelectrolytes and inject and/or extract ions from the terminals. In the active mode (left, VEC=10ACh transport from emitter to collector. In the off-mode (right, VEC=10 V and VEB=−1 V), th(B) Intracellular Ca2+ recording of ACh stimulated SH-SY5Y cells cultured on the collector terRef. [38] and figure B is reproduced with permission from Ref. [37], courtesy of pnas.org.

types of IBJTs promise for the development of addressable X–Y ma-trixes with controlled release of both positively and negativelycharged ions and biomolecules. We foresee that such matrix systemwill be useful when studying cell signaling in neuronal networks,i.e. circuits of connected neurons where information is exchangedvia electrical and chemical signals.

4. Towards artificial nerve cells for in vivo applications

There is a current trend amongst researchers and in the medicaltechnology industry to generate drug delivery systems and machine-to-brain interfaces that eventually will benefit the patient. As demon-strated above, the OEIP delivery technology is based on several featuresimportant to consider when designing devices for in vivo applications.This includes the following: i) the ability to turn delivery on and offusing the electronic control; ii) bio-molecules are delivered in theabsence of liquid flow; iii) delivery is obtained with no burst release;iv) delivery is obtained with minimal leakage.

In our first attempt to demonstrate the use of the OEIP as a novel“machine-to-brain” interface, in which computer-generated electronicinput signals are transformed to the release of chemical substances fordirect modulation of mammalian senses, we used the auditory system.The cochlea provides relatively easy access to the sensory organ andallows a direct communication pathway between the device and thebrain. Studies of the auditory system are commonly performed usingthe guinea pig as model system, since the structure of the cochlea andthe range of hearing frequencies are very similar to that of humans.Hence, the auditory system of the guinea pig has been used extensivelyin validation of local delivery systems as the effect on the auditory nervecan bemonitored [40,41]. However, local delivery to the cochlea is chal-lenging, as the delicate mechano-sensitive hair cells in the smallfluid-filled structure can easily be damaged [42].

Auditory transduction is the process where a sequence of eventstransforms sound waves in the air into electrical impulses interpretedby the brain. Sound waves enter the ear and are transported along thehearing duct where they set the cone shaped eardrum in vibration.From the eardrum, the vibrations are transduced over the auditory ossi-cles into the snail shell shaped cochlea. In the cochlea, auditory sensorycells responsible for generation of the nerve impulses are sent to thebrain. There are two types of auditory sensory cells, the inner andouter hair cells. The outer hair cells (OHCs) amplify and tune the signalwhile inner hair cells (IHCs) are responsible for transferring the electricimpulses to the primary auditory neurons, the spiral ganglion neurons

of a neutral polymer gel electrolyte. The conductive PEODT:PSS electrodes are covered byand VEB=4 V), the base supplies the junction with Cl−. Increased conductivity results ine base depletes the junction of Cl− and ACh delivery stops due to decreased conductivity.minal. Turning the base on/off regulates ACh-delivery. Illustration in A is modified from

Fig. 8. (A) The syringe-like encapsulated OEIP. (B) Illustration of the cross section of theorgan of Corti in the cochlea and the devicemounted on the RWM. (C) Measuring the au-ditory brain stem responses the hearing attenuation is shown as a function of recordingfrequency upon delivery of Glu (blue) and H+ (yellow) at 15 min (dashed bars) and60 min (solid bars). (D) Histological analyses demonstrate the control (left) and dendritedamage (right) indicated by asterisks (*). Figure A reproduced with permission from Ref.[53], courtesy of GIT Verlag, and B–D is reproduced from Ref. [30].

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(SGNs). As the basalmembrane vibrates, stereocilia “hairs” on the apicalpole of the hair cell are deflected against the tectorialmembrane closelycovering the hair cells. The deflection causes opening of mechano-sensitive ion channels and the influx of K+, which depolarizes the cell.The alteredmembrane potential results in opening of VOCCs and subse-quent Ca2+ influx causes transmitter release. Glutamate is the primaryneurotransmitter for the IHC and when released it stimulates dendriteson the postsynaptic cell, the SGN [43]. The afferent SGN receives thesignal and transfers it via the auditory nerve, to the cochlear nuclei inthe brain stem. Exposure to traumatic noise will cause overstimulationof IHCs and an excessive release of glutamate at the IHCs' afferentsynapses. Glutamate overstimulation at the dendrites of postsynapticcells will in turn cause massive entry of cations and water, subsequentdendrite swelling and loss of contact between IHCs and SGNs dendrites[44]. The excitotoxic effect of glutamate disturbs the Ca2+ homeostasisin the SGN and can lead to cell damage and death.

4.1. Design and functionality of the in vivo OEIP

To enable the OEIP to work as a machine-to-brain interface, the pla-nar device was re-designed to a more flexible, tube-like shape with apotential to be surgically implanted. The planar device was separatedinto two encapsulated electrode-electrolyte systems, the source andtarget compartments (anode respective cathode) (Fig. 8A). This allowedfilling of the source compartment (anode) with the substance to bedelivered and the cathode system with a NaCl electrolyte. By cuttingthe over-oxidized and electronically insulated ion channel in half, thetip from the source and target electrodes (outlet/inlet) comes in directcontact with the target system. When used in vitro, the target systemcan be cell media or a buffer, while in vivo the target system can beany type of bio-fluid. To complete the electrochemical circuit, cationsare extracted from the bio-fluid into the target compartment (cathode).

The functionality of the new designwas first tested in vitro. As gluta-mate was the signal substance of choice to be used in the in vivo exper-iment, the biological activity of OEIP-transported Glu was evaluated onprimary cultures of astrocytes known to express the Glu-receptors. Theworking principle is the same as in the planar deviceswhere the voltagedrop occurs primarily across the channel, resulting in negligible electricfields in the target system. Hence, the cells will not be triggered by theapplied potential or electric field. When transported Glu reach thetip of the device, it is released into the target electrolyte, from whereions are spreading by diffusion to adjacent astrocytes in culture. Glu-responsive receptors are activated and results in opening of Ca2+ chan-nels promoting a robust Ca2+ influx into the cells. By transporting Gluto the cells while at the same time monitoring the real-time Ca2+

responseswedemonstrated how the tube-like device could be operatedin vitro [30].

4.2. Modulation of mammalian senses

The tube-like design of the OEIP was next utilized for non-invasivedelivery of Glu to the perilymph in the cochlea. The small volume ofperilymph poses a challenge, since liquid and pressure-induced tissuedamage needs to be minimized. Hence, the tip of the syringe-likeOEIP was mounted at the bone beside the round window membrane(RWM) of the cochlea (Fig. 8B). Using the RWM as a port of diffusiveentry into the cochlea, continuous delivery of Glu, was performed at aconstant voltage for 1 h [30]. An exceedingly high concentration ofGlu is known to result in swollen dendrites of the SGNs and loss ofcontact with the inner hair cells [44], a mechanism previously utilizedin validation of local delivery systems as the effect of the deliveredsubstance can be monitored as reduced hearing sensitivity [40,41].By monitoring the brain's ability to perceive sound of specific fre-quencies, so called auditory brainstem responses (ABR), the hearingsensitivity was assessed before and after Glu-delivery with the OEIP.During the 1 h exposure, ABR measurements showed that the animal

suffered from significant loss of higher frequencies at the base whereGlu enters via the RWM, whereas the control group exposed to H+

showed no change in hearing sensitivity (Fig. 8C). In addition toconfirming the excitotoxic effect of Glu, this experiment demonstrat-ed the inert nature of the OEIP in vivo delivery system, meaning thatthe device application and molecular transport itself was not damag-ing. After completion of the experiment, histological analysis of thecochlea confirmed the cellular and molecular details of the mecha-nism for auditory impact (Fig. 8D).

Collectively, these experiments demonstrate the usability of theOEIP technology as a machine-to-brain interface. As electrical inputsignals are translated to delivery of a specific number of chemicalmessengers, specific targeting is achieved, as only the type of neuronthat expresses the cognate receptors will be activated. This is in con-trast to systems based on electrical stimulation, i.e. deep brain stimu-lation, where any excitable cell type in the vicinity of the electrodewill be activated.

5. Concluding remarks and perspectives

This review describes the interdisciplinary efforts, aiming to developnovel communication interfaces between electronic components andbiological systems. Utilizing the electronic and ionic properties of conju-gated polymers, a toolbox of organic bioelectronic devices has been de-veloped that can be used individually or in combinations to regulate cell

Table 3Capabilities of the OEIP.

OEIP capabilities: References:

1. Non flow-based delivery system with diffusive release. [28]2. Electronic addressing with on/off control. [28]3. Easy integration of the soft and flexible organic material in biologicalsystems.

[28]

4. Delivery of the cations H+, K+ and Ca2+. [28,29]5. Delivery of single stimuli to cells in vitro. [28]6. Quantification of total amount of delivered ions and calculations oftransport efficiencies.

[28]

7. Generation of temporal concentration gradients and oscillations. [29,34]8. Transport of ions and neurotransmitters. [30,34]9. Modulation of the amplitude of Ca2+ responses by regulatingapplied voltage or time.

[34]

10. Generation of Ca2+ oscillations by regulating applied time. [34]11. Regulation of ion flows in electrophoretic transistors. [37]12. Delivery of single stimuli to cells in vivo. [30]

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signaling in vitro and in vivo. Table 3 provides a summary of the uniquefeatures of the planar and tube-like OEIP devices, and the IBJT. Theorganic bioelectronics research field is advancing at a high pace. Thisincludes further development of material properties, design of devices,and expansion of the molecular transport repertoire. This collectiveeffort will aid in making organic bioelectronics devices useful for anumber of applications in vitro. Also, one can foresee a number oftherapeutic areas that would benefit from this technology, as itestablishes itself as a candidate for the next generation of implantablebiomedical delivery devices.

Acknowledgements

We thank all our partners for fruitful collaborations in these highlyinterdisciplinary research projects. Research in the ARD laboratory issupported by the Swedish Medical Nanoscience Center, the SwedishFoundation for Strategic Research (OBOE Strategic Research Centerfor Organic Bioelectronics) and The Swedish Governmental Agencyfor Innovation Systems (VINNOVA).

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