Conductive Polymers to Modulate the Post-Stroke Neural Environment

Abstract

Despite the prevalence of stroke, therapies to augment recovery remain limited. Here we focus on the use of conductive polymers for cell delivery, drug release, and electrical stimulation to optimize the post-stroke environment for neural recovery. Conductive polymers and their interactions with in vitro and in vivo neural systems are explored. The ability to continuously modify the neural environment utilizing conductive polymers provides applications in directing stem cell differentiation and increasing neural repair. This exciting class of polymers offers new approaches to optimizing the post-stroke brain to improve functional recovery.

1. Introduction

Stroke remains a devastating disease for patients and their caregivers. Stroke is the 2^nd leading cause of death in the world with societal costs of over $100 billion annually in America alone (1, 2). While acute ischemic therapies aimed at clot removal have rapidly advanced in recent years, methods to improve brain tissue recovery in the subacute and chronic windows remain in the realm of preclinical and clinical research. Better options to enhance functional recovery outside of the acute stroke window are required.

Biomaterials remain an attractive approach to interact with the neural environment following stroke. The ability to deliver drugs, cellular therapeutics, and manipulate the post-stroke milieu are advantages of their use. Various non-conductive biomaterials have been used to interact with the nervous system to deliver drugs or optimize cellular therapies (3–7). However, these are often limited by the inability to modulate the polymer after implantation. Given the electrical, mechanical, and chemical interactions of the nervous system, conductive polymers are an especially appealing biomaterial that provides a platform for continuous interaction with recovering neural tissue. In this review, we will summarize the complex conditions following ischemic stroke including the current treatment options and explore the opportunities that conductive polymers provide to augment healing after stroke.

2. Pathophysiology of Stroke

2.1. Acute Injury

An ischemic stroke is caused by an occlusion of a blood vessel supplying the brain, most commonly from a blood clot. The inadequate supply of blood resulting from this occlusion is referred to as ischemia. As the metabolic demand of the brain surpasses the concentration of oxygen and glucose supplied to the tissue during a stroke, a complex series of events elicits injury pathways and cell death. The neural cell membranes become unstable in the ischemic environment, and an intracellular influx of calcium triggers excitotoxicity (8). This rapid influx of calcium largely through glutamatergic pathways leads to mitochondrial dysfunction which can trigger cell death pathways such as apoptosis. Also in the ischemic brain, necrotic cell death occurs and increases the inflammatory environment (9). These processes trigger free radical release which can worsen cellular toxicity and further prevent recovery (10). Pursuing strategies that limit these injury pathways have been a major target of stroke therapeutics.

2.2. Post-stroke Inflammatory Response

The initial central nervous system (CNS) inflammatory response is composed of microglia navigating to the ischemic injury. Subsequently, the breakdown of the normal connections between the glia, neurons, and endothelial cells forming the neurovascular bundle results in loss of integrity of the blood brain barrier (11). This breakdown exposes the neural tissue to systemic inflammatory cells which are generally prevented from entering the CNS. The introduction of the systemic innate and adaptive immune responses causes neural damage in the acute and sub-acute periods after stroke (12). Conversely, many of these processes likely contribute to the remodeling and clearance that is necessary for eventual repair of the damaged tissue.

Recent work has also indicated that a chronic immune response may contribute to post-stroke cognitive changes and outcomes (13). Further understanding of the early and late inflammatory changes after stroke will help in understanding the body’s response following stroke. This improved insight will provide further targets for stroke interventions.

2.3. Neural Repair

Subsequent to the acute injury, multiple pathways for repair occur, including neurogenesis and angiogenesis. Removing damaged synapses, remodeling new synaptic connections and reforming the networks of dendritic spines help to restore some function after stroke. Inflammatory and structural remodeling processes that originally may have contributed to the injury also clear the way for recovery to occur (14). The electrical balance of the brain is also altered after stroke. The restoration of this balance is important for recovery with specific types of inhibitory synaptic signaling playing key roles in neural repair (15, 16). Conductive materials that can help control the electrical environment following stroke provide a powerful tool to enhance recovery. Understanding these processes and forming materials to increase the healing pathways is an exciting area of research.

With angiogenesis, new vessels are formed to restore circulation to the damaged tissue (17, 18). Often, these new vessels are not developed sufficiently to support blood flow but may serve as a structure to allow for migration of cells to help with the neural remodeling (19). As the vasculature becomes more mature, portions of the penumbral tissue consisting of viable tissue in the peri-infarct region are able to recover.

Endogenous mechanisms of repair include the generation of endogenous stem cells. Endogenous stem cell production in the subventricular zone is increased following stroke (20, 21). These endogenous stem cells have a neural fate and migrate to the site of injury to enhance repair. Electrical stimulation has been shown to increase the production of endogenous stem cells and chemical factors such as granulocyte-colony stimulating factor have also been targeted to enhance this endogenous repair pathway (22, 23). Treatments that amplify the neural recovery pathways would have great impact on improving the lives of stroke survivors.

3. Treatment Options for Stroke

3.1. Clot Removal

To date, the most effective therapies for ischemic stroke are aimed at removing the clot from the occluded vessel to restore blood flow. Stroke patients who present within 4.5 hours of the last time they were observed to be normal are generally eligible to receive intravenous tissue plasminogen activator (tPA) for chemical thrombolysis (24). After this time window, the bleeding risks outweigh the benefit, but mechanical thrombectomy procedures have been developed to physically remove the blood clot. Mechanical thrombectomy has even proven beneficial up to 24 hours after symptom onset when in conjugation with advanced imaging techniques that can select patients with penumbral tissue that remains salvageable (25, 26). These advances have dramatically increased the number of patients who are eligible for acute treatment after stroke. The wider use of acute interventions will likely create a greater number of stroke survivors with lifestyle-limiting deficits, and thus increase the need for improved stroke therapy options to recover from these deficits.

3.2. Neuroprotection

Many of the initial attempts to improve function after stroke centered on limiting the initial damage with the use of neuroprotective agents (27, 28). Unfortunately, many of the agents that demonstrated great effectiveness at improving functional outcomes in the lab failed to translate to benefit patients. These failures led to re-evaluation of pre-clinical methods and ultimately improved experimental models to more closely mimic the clinical setting. Advanced imaging selection may also provide an avenue to better select patients with more viable tissue for treatment and improve the results of neuroprotective trials.

Neuroprotective strategies that aim to effect multiple pathways may also yield improved results compared to targeting a single pathway. Hypothermia which affects numerous injury mechanisms improves outcomes in global ischemia in children and adults (29, 30). Avenues of research continue to evaluate protective methods that target multiple deleterious pathways for more focal ischemic damage in adults.

3.3. Neural Recovery

Current stroke recovery therapy is largely based upon physical and occupational rehabilitation. No FDA approved medical therapy exists at this time to improve healing following stroke. Fluoxetine combined with standard therapy has been shown to improve motor recovery in a small trial of stroke patients (31). The use of robotic devices to assist motor recovery and wearable electronics to guide therapy are being investigated to help guide more effective therapy. Electrical stimulation with transcranial direct stimulation (tDCS) and transcranial magnetic stimulation (TMS) have shown promise in preclinical experiments and small clinical trials (32–34). Although they have yet to show lasting efficacy in the clinic, continued efforts are an exciting area of exploration.

Cell-based therapies have emerged as an intriguing area of research. Multiple early phase clinical trials have indicated that stem cell therapy may be a viable treatment to improve recovery in the sub-acute and chronic time periods (35, 36). Numerous pre-clinical and clinical studies using induced pluripotent stem cells (stem cells re-programmed from a patient’s own skin), mesenchymal stem cells, and embryonic derived stem cells are promising (36–42). While a few stem cells may integrate into the damaged tissue depending on the stem cell types, many of the studies to date suggest that a large amount of the therapeutic effect of the stem cells relies upon the trophic factors that they release (43–45).

Many questions remain as to the best stem cell type and the optimal delivery method. The ideal route of delivery whether it be an intracranial, intravenous, or intranasal continues to be explored. The timing of delivery is another question that must be answered to optimize stem cell treatments. The exact mechanism of action of each stem cell type needs to be better understood to more effectively engineer treatment strategies.

As the stroke and post-stroke environment are more fully understood, biomaterials offer a unique tool to interact and optimize the recovery pathways. Delivery of cells or therapeutic agents can be timed to limit injurious pathways or strengthen mechanisms of recovery. With the ability to provide a biocompatible cell scaffold and to continuously interact electrically with stem cells and the surrounding neural environment, conductive polymers offer a compelling platform to address many of the challenges of stroke recovery.

The ultimate goal of utilizing conductive polymers for neural tissue engineering is to be able to mimic the endogenous tissue environment found in a healthy brain or manipulate neural activity in the recovering brain (46). Various biophysical signals including chemical, mechanical, material-based, and electrical cues govern the proliferation and maintenance of tissue functionality (47–49). Exogenous signals such as chemical and mechanical topography have been substantially investigated, and these signals are well-established tools in the in vitro creation of tissue mimics (50, 51).

4. Conductive Polymers to Treat Stroke

4.1. Conductive Polymers

Electrically conductive biomaterials consisting of carbon nanotubes, graphene, and inorganic particles (i.e. gold/silver nanomaterials) have been investigated in neural tissue engineering applications due to their electrical excitability (52–55). However, those biomaterials are not biodegradable and, in some cases, cause long-term in vivo toxicity (56, 57). Conductive polymers including polypyrrole (PPy), polyaniline (PANi), and poly(3,4-ethylenedioxythiophene) (PEDT, PEDOT) were introduced as alternatives and merged the positive aspects of conductive biomaterials and inorganic materials (58, 59).

These conductive polymers share several properties. They conduct charge by free movement of electrons between atoms arranged by a series of alternating single and double bonds with overlapping pi-bonds (Figure 1). A doping process introduces the charge carriers into the polymer, creating the polymers’ conductive nature. Different doping processes exist, and the conductivity can be further modified by the amount of dopant (60, 61). By designing and selecting dopants in the solution, the structural and surface properties of the conductive polymer can be controlled.

Figure 1.

Chemical structures of conductive polymers. PPy: Polypyrrole; PEDOT: Poly (3,4-ethylene dioxythiophene); PANi: Polyaniline.

PPy has been widely utilized for neural tissue engineering due to its reasonable conductivity (1-75 S/m) under physiological conditions (43, 62). In addition, it has shown great biocompatibility in both in vitro and in vivo systems as well as the ability to manipulate tissue interactions (63–65). For example, a PPy thin film coated with fibronectin was utilized to support aortic endothelial cell growth (66). In contrast, cell growth and DNA synthesis were inhibited when a PPy thin film without fibronectin modification was used. This data suggests that conductive polymers introduce an external means to modulate the shape and function of adherent cells. PEDOT has gained recent use given its ability to form highly stretchable, conductive polymers (67). This provides the means to adapt the conductive polymer to the changing biological surface. PANI is another conductive polymer providing the flexibility to interact with the CNS electrically (68).

4.2. Impact of Electrical Stimulation

Recently, electrical stimulation has become an active area of cell biology and tissue engineering research due to its role in the functioning of all living organisms (Table 1). Electrical signals play a part in controlling cellular functions such as gene expression, proliferation, and intracellular signaling (69, 70). Additionally, electrical activity for delivery of pharmaceuticals such as small molecules, genes, and proteins can control the delivery of therapeutics to achieve the repair or reorganization of tissue growth (71). By external electrical stimulation, we should be able to gain greater control over nerve cell growth, proliferation, and maintenance (72). Furthermore, by applying electrical stimulation to tissue engineered-products utilizing PEDOT, improvements may be possible to support neural regeneration, to enhance neural specific differentiation, and to increase the length of neurite outgrowth (73).

Table 1.

Various conductive scaffolds and electrical stimulation for neural tissue engineering

Conductive Scaffolds	Stimulation Pattern	Duration	Type of Cells	Major Outcomes	Ref.
PPy	DC: 100 mV	2 hr	PC12	Neurite outgrowth	91
PPy/PLGA	DC: 10 mV	2 hr	PC12	Neurite outgrowth	92
PPy	AC: 1 V/1kHz	1 hr	NPCs	Stroke recovery	43
PPy	AC: 800 mV/100 Hz	1 hr	NPCs	Modulation of trophic factors	62

PPy: Polypyrrole; PLGA: Poly lactic-co-glycolic acid; DC: Direct current; AC: Alternative current; PC12: Pheochromocytoma; NPCs: Neural progenitor cells

Tissue or cells are the main source of in vivo electricity. The nervous system communicates via a sophisticated combination of chemical and electrical signals (74). During neural development, these signals guide stem cell fate and circuit formation. In embryonic cells, for example, voltage-gated sodium and calcium channels generating electrical signals are critical for the development of neural precursors and differentiating neurons (75). Disruption of these signals in embryos has been found to cause serious defects including the absence of the cranium, a malformed head, and the loss of eyes. In addition, disrupting these electrical currents on partially amputated Xenopus tails played a critical role in regeneration.

Because of the effect of electrical stimulation on the body, a wide range of approaches have been utilized in implantable and external stimulation devices (76–78). In fact, electrical stimulation has been applied across a variety of conditions including bone growth support, chronic wound healing system, and muscle stimulation in paralyzed patients (79–82).

4.3. The Methods of Electrical Stimulation

4.3.1. Direct Stimulation

Transcranial direct current stimulation was able to improve rehabilitation outcomes after stroke by augmenting brain plasticity (83). Direct stimulation delivers electrical signals through an electrode that is in contact with the cell culture system. Mesenchymal stem cells were widely studied with direct stimulation due to their versatile differentiation lineages such as osteogenic, endothelial, and neural cells (84, 85). Direct stimulation can change cell behavior such as migration, proliferation and differentiation of bone marrow derived mesenchymal stem cells (MSCs) (86). Direct stimulation of 100 mV/mm was applied to change osteogenic gene expressions in MSCs (Runx2, Osteopontin, and Col1A2) at day 7. Although this approach is straightforward and easy to design, it causes several drawbacks such as toxicity associated with pH changes and generating reactive oxygen species in the culture medium. Moreover, the efficacy of the stimulation is heavily dependent on placement of the electrodes. Current flow occurs near the anode or the cathode electrodes, which further hinder the use of this method.

4.3.2. Indirect Stimulation

Indirect stimulation for therapeutic devices and in vitro experimental setup avoids the difficulties associated with direct stimulation. A homogenous electromagnetic field (EMF) is a physical energy field which plays a critical role in numerous biological processes (87, 88). This generates small magnitude currents and potentials in the presence of metal substrates. It delivers the stimulation in the proximity of the targeted cells adjacent to metal substrates rather than simply supplying the charge via electrodes. Taking advantage of this non-invasive and controllable technique, the therapeutic utilization of EMF in regenerative medicine has emerged. Electromagnetized gold nanoparticles placed in Helmholtz coils augmented the efficacy of direct conversion of somatic fibroblasts into pluripotent stem cells and induced dopaminergic neurons in vitro (89, 90). A subsequent in vivo study in Parkinson’s disease models revealed that electromagnetized gold nanoparticles accelerated in vivo dopaminergic neuron reprogramming, resulting in alleviation of the disease symptoms. This study demonstrates a proof of concept for indirect stimulation as a safe therapeutic strategy for regenerative medicine.

4.3.3. Electrical Stimulation through Conductive Polymer Scaffolds

Evolving from the approaches described above, conductive polymer scaffolds have emerged as a new means to apply electrical stimulation to biological systems. PC-12 cells cultured on PPy films with applied electrical stimulation showed a significant increase in neurite outgrowth (91). Furthermore, PPy coated on PLGA nanofibers via in situ polymerization was utilized to induce the synergistic effect of different cues on neurite outgrowth (92). Recent publications demonstrate that a PPy scaffold provides a method to modulate intracellular activity of neural progenitor cells (NPCs) (43, 62) (Figure 2). NPCs exposed to evenly distributed, well-controlled electrical stimuli via the PPy scaffold were able to produce high concentrations of paracrine factors such as VEGFA and BDNF. By using these conductive polymer scaffolds as a source of electrical stimulation, mouse retinal progenitor cells (mRPC) cultured on the scaffold can be directed to retinal differentiation (93). Further hybrid surfaces using PEDOT and chitosan modify neural cell differentiation (94). PANI-based scaffolds also optimize stem cells for neural applications (95, 96). These studies indicate a role of electrical stimulation through conductive polymers for biological applications and to control stem/progenitor cells differentiation toward neural fates.

Figure 2.

Illustration of cell culture system using PPy scaffold. NPCs were plated on PPy scaffold and cells were electrically modulated by stimulation (AC: 800 mV; 100 Hz). a. Assembly of cell culture system using conductive scaffold. b. NPCs were cultured on the surface of PPy scaffold and cells were stimulated by waveform generator. c. Picture of PPy cell culture system. d. Expression of BDNF from NPCs were modulated by electrical stimulation (this figure courtesy of Oh et al, 2018 (62)).

4.4. Application of Conductive Polymers

4.4.1. Conductive Polymers for Neural Probes

The application of conductive polymers as neural probes holds great promise (97). Neural probes facilitate the functional stimulation and recording of neuronal networks in the brain (98). However, the development of a flexible, stable and biocompatible probes is necessary for longterm implantations (99). Surface modification of electrodes has been introduced to improve longterm stability and functionality with PEDOT and other conductive polymers (100–102). Neural probes functionally coated with PPy-nano peptide CDPGYIGSR enabled cell adhesion and neurite extension in vivo (99). In addition, an in vitro cell sensing system using PPy/RGD composite film has been developed to monitor cell behaviors and cytotoxicity (103). As our understanding of electrical modulation to help with neural recovery continues to advance, the advantages of polymeric based-systems for this application offer many advantages over non-polymeric methods, and conductive materials can begin to be applied to regenerative applications such as enhancing stroke repair.

4.4.2. Conductive Polymers as Drug Delivery Systems for Stroke Recovery

Electrically tunable drug delivery systems allow for release profiles that can be tailored to match pathophysiologic processes. A previous study demonstrated that PPy could be formed in the presence of biotin as a dopant (104, 105). Electrical stimulation accelerated the reduction of the PPy backbone, which triggered the release of the biotin and the attached payload. Biotinylated NGF released from the PPy film was still active, and it promoted neurite outgrowth of PC-12 cells. A PPy film doped with graphene oxide nanosheets is able to electrically control the anti-inflammatory drug (dexamethasone, DEX) release. This approach creates the opportunity to address dosing needs that may change over the course of treatment to enhance recovery following an neurologic injury (106).

4.4.3. Conductive Polymers as Cell Delivery System for Stroke Recovery

Conductive polymers offer a unique approach to interact with stem cells and modulate their intracellular properties. Biocompatible hydrogels offer protection of cells implanted into the harsh stroke milieu and augment survival (107, 108). Because of their non-conductive characteristic, the microniche for the implanted cells cannot be controlled externally after implantation. On the other hand, conductive polymers offer a novel method for protecting transplanted stem cells and continuously interacting with cells through electrical stimulation.

Conductive polymer’s potential to be used as growth substrates that direct human stem/progenitor cell fate is intriguing for recovery applications. It has been noted that endogenous electrical fields generated by neuronal networks are highly associated with fetal development, as well as cell differentiation fate. Electrical stimulation of a PPy film induced human neural stem cells (hNSCs) to differentiate into predominantly Tuj1-expressing neurons and formed nodes or clusters of neurons joined by neurite networks (109). In addition, hNSCs differentiation was augmented by electrical stimulation using a cross-linked PEDOT substrate (73). These studies indicated that conductive polymers support exogenous cell culture, and they can be furthermore modulated by electrical stimulation due to their conductivity.

Neural stem/progenitor cells can be also delivered using conductive polymer for stroke recovery. Previously, it has been reported that an electrically conductive scaffold made of PPy allows for in vitro electrical stimulation and subsequent implantation of human hNPCs onto the peri-infarct cortex (43, 62). A short period of electrical stimulation prior to implantation changed gene expression in the hNPCs to improve functional recovery by modulating VEGF-A production. A recent study also demonstrated that directional currents in the brain mobilizes migration of transplanted hNPCs (110). These studies revealed that electrical stimulation provides a potential strategy to manipulate, facilitate, and guide stem cell therapy for stroke.

5. Future Perspectives on Conductive Polymers to Treat Stroke

Treatment of stroke and other neural injuries using conductive polymers is beginning to garner interest. Advances in chemistry, biology, and material sciences have implications for the use of advanced, biocompatible, and stimuli-responsive conductive scaffolds (Figure 3). Conductive scaffolds are ideal for delivering electrical signals to the brain. They are well-suited for combination therapy with stem cells targeted at increasing the brain’s regenerative capacity. Studies have shown that electrical stimulation of stem cells modifies their characteristics and that the electrical signaling augments their ability to improve recovery. One important mechanism is the increased production of paracrine factors after the electrical stimulation. The relationship between the stem cells and the conductive polymers and their effect on the injured brain offer exciting prospects for future clinical therapeutics.

Figure 3.

Schematic representation of electrical stimulation using a conductive polymer scaffold. Various stimulations including direct, inductive, and conductive scaffolds regulate cells resulting in increase in trophic factors, cell proliferation, and controlling stem cell differentiation.

6. Conclusions

Given the electrical, mechanical, and chemical interactions of the nervous system, conductive polymers are an especially appealing biomaterial that provide a platform for continuous interaction with recovering neural tissue. Conductive polymers offer a unique method to modulate the post-stroke environment through stem cell optimization, delivering electrical stimuli, and precisely controlling chemical therapeutics to improve recovery.

Highlights.

• Conductive polymers possess the ability to electrically interact with neural tissue

• After stroke, conductive polymers can shape the electrical environment

• Combined with stem cells, conductive polymers affect stroke recovery pathways