Protein adsorption and peroxidation of rat retinas under stimulation of a neural probe coated with polyaniline
Introduction
Current research on microfabrication technology for implantable neural probes has been maturely developed. Processes for fabricating microelectrodes from glass, gold, platinum (Pt), silicon and various polymers are already established [1], [2], [3], [4]. However, there still have two factors inhibiting their clinical application. (1) Implantation of stimulating electrodes can produce soft tissue wounds with inflammation, which is a host response to destroy, dilute or wall off the injurious agents and injured tissue. If inflammation persists for more than 2 weeks, it is classified as chronic [5]. The localized defense mechanism generally leads to encapsulation by microglia, astrocytes, endothelia, fibroblasts, etc., and increases the impedance across the electrode to limit the functionality. As a result, a higher input energy could be required and ultimately lead to device failure [6]. Observation of the encapsulation of micromachined silicon devices of different size and cross-section was carried out by Turner et al. [7] and Szarowski et al. [8]. This showed a clear GFAP-positive astrocytes sheath around the insertion site after 4 weeks and an increased number of GFAP-positive stellate astrocytes. This prolonged response has been found to be independent of the device size, geometry and surface roughness, and revealed that implant coating materials with properties of reducing protein adsorption and cell attachment may be critical to long-term functionality. (2) Another factor is the function degradation caused by the corrosion of the noble metal electrode. Pt and its alloys have demonstrated acceptable electrode behavior because of the inertness, biocompatibility and the ability of reducing charge gathering from electrical stimulation. However, Pt electrodes using direct currents to stimulate in the moist environment functioned with far less electrical impedance and toughness than in body surfaces in contact with dry air. In the moist oral cavity, Pt electrodes produced a necrotic tissue response on non-keratinizing tissue even at relatively low current densities of <100 μA cm−2 [9]. For neural and neuromuscular prostheses, where the current densities reach 0.1–0.6 A cm−2, there can be degradation of the noble metal electrodes and problems with biocompatibility under chronic stimulation [5].
Current work moves towards the improvement of the tissue–electrode interfaces. Approaches in improving the implant–tissue interface include surface modification by electrochemical polymerization and covalent immobilization of a biocompatible coating [10]. Ideally, the electrode material would be bioactive in encouraging neurite growth around the electrode as well as preventing the formation of a fibrous capsule. The ability to achieve this is very limited when using metals, but using polymers allows for enhanced bioactivity [6]. There are a few polymers with dual properties of biocompatibility and electrical conductivity, such as polypyrrole and polyaniline (PANi) [11], [12]. They exhibited metal-like conductivity and were used as coatings on electrodes rather than as free standing material, owing to their very fragile nature [13], [14]. PANi is one of the most promising electrically conductive polymers and has been widely investigated because of its unique properties, including controllable electrical conductivity, environmental stability, and optical and electro-optical properties [12], [15]. It is possible to achieve relatively high conductivities within the appropriate fabrication technique. By electrosynthesis, PANi can be coated on inert electrodes such as gold, Pt and aluminium alloy [16]. Many reports present the production of PANi coating by casting [17], [18]. Previous studies prepared the synthesized PANi films through modified polymerization. The synthesized PANi films were composed of nanoparticles at 30–50 nm and had much better cell behavior with PC-12 pheochromocytoma cells, such as attachment and proliferation, while the casting PANi film had no such cell behaviour and structure on the nanoscale [19]. Besides PC-12, more evidence showed the ability of PANi and its variants to support cell growth, especially neural cells [20]. Also, it supports the adhesion and proliferation of H9c2 cardiac myoblasts and enhances in vitro neurite extension [21]. A large surface/volume area, controllable surface morphology, wettability, high capacitance and the ability to remain conductive over long periods afford benefits for neural probes. These advances encourage the development of optimized neural electrodes.
Nevertheless, neural electrodes can cause damage to the nerve [22]: first, by the surgical implantation procedure, depending on how much the nerve is separated from the neighbouring structures, accompanied by traumatic interference with the vascular supply. The surgical procedure can damage neighbouring arterial beds and possibly the internal capillary network, as well as the nerve fibres and fascicles, which results in various degrees of functional impairment and intra- or extraneural fibrosis, which interferes with intraneural blood flow [23]. Another potential of damage can be the electrode itself, which produced mechanical restriction on the nerve, but such injury depends on the degree of tissue–electrode contact and its duration. Evidence suggests that overstimulation may either modulate vital ionic and protein gradients, leading to cell death, or generate cytotoxic and apoptotic products by Faradaic reactions at the electrode–tissue interface [24], [25]. Besides physical injury, dissolution of metal electrodes, excitotoxicity and dielectric breakdown of the cell membrane also produce neural damage in the cerebral cortex [26]. Most studies examining chronic stimulation injury in the retina have relied on anatomical methods of assessing cytotoxic damage [27], [28].
Retina is the eye neurosensorial tissue and is very rich in polyunsaturated fatty acids (PUFA). It is an excellent model for studying electrical stimulation-induced peroxidation because of its high susceptibility to oxidative damage due to the high content of PUFA. The reactive oxygen species induce lipid peroxyl radical formation and initiate lipid peroxidation, which can injure the retina, especially the membranes that play important roles in visual function [29]. Furthermore, biomolecules such as proteins or amino lipids can be covalently modified by lipid decomposition products, including forming Schiff bases with aldehydes or/and activating membrane-bound enzymes [30], [31], [32].
Tissue lipid peroxidation during electrical stimulation was detected by Devyatkina et al. [33]. An increase in lipid peroxidation was found to be raised during stimulation. Lipid peroxidation is a complex process which refers to the oxidative degradation of lipids. The process is a self-perpetuating reaction, called a “chain reaction mechanism”, involved in the abstraction of a hydrogen atom from a methylene carbon to form a lipid hydroperoxide on PUFA, and rearrangement of the double bonds to form an alkyl radical with a conjugated diene. It continues until meeting termination by free radical scavengers or other free radicals. Breakdown of lipid hydroperoxides leads to the formation of various molecular species and causes cytotoxicity [34]. In ocular tissue, it is correlated to a wide range of disease processes, including cataractogenesis and retinopathy of prematurity [35], [36].
In this paper, retinal homogenate was used to evaluate tissue damage from the viewpoint of biochemistry from both phospholipid (PL) peroxidation and protein denaturalization under electrical stimulation of the uncoated Pt electrode with or without PANi coating. The PANi-coated Pt electrode, with a regular and compact nano-PANi-particle surface, was fabricated by in situ polymerization of PANi onto the uncoated Pt electrode surface. Dynamic adsorption of retinal fragments was detected by atomic force microscopy/scanning electron microscopy (AFM/SEM) and was quantified by micro BCA protein assay; peroxidation of rat retinas induced by electrical stimulation from the PANi-coated Pt electrode was evaluated by quantifying the change of conjugated dienes and phospholipid hydroperoxides (PLOOH), especially phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH); changes of retinal proteins from electrode surfaces were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Long-term in vitro electrical stimulation of the uncoated Pt electrode with or without PANi coating was also observed by SEM and UV–visible spectrophotometry.
Section snippets
Animals and materials
Sprague–Dawley (SD) rats (220 ± 30 g) were purchased from the Animal Center of Fudan University, and the study design was approved by the Ethical Committee of Animal Experiments of Shanghai Jiao Tong University.
Aniline was purchased from Aldrich (St Louis, MO). HCLO4 and ammonium peroxydisulfate ((NH4)2S2O8) were purchased from SCRC (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and used as received. A micro BCA protein assay reagent kit and coomassie blue dye were purchased from Pierce
Preparation of the PANi-coated Pt electrode
Conducting polymers (CP) were first reported by Heeger et al. [47] in 1977. CP are a class of functional polymers with a highly conjugated polymer chain, which can be assigned reversible chemical, electrochemical and physical properties and controlled by a doping/de-doping process. Polyheterocycles, such as PANi, PPy, polythiophene and poly(3,4-ethylenediox-ythiophene) (PEDOT) developed in the 1980s, exhibit good stability, conductivity and ease of synthesis [48]. Nowadays, CP with
Conclusions
By modifying the fabrication method, the present authors successfully polymerized in situ PANi with a regular and compact nanostructure 20–40 nm in diameter on the uncoated Pt electrode surface. The PANi coating exhibited several excellent properties, including inactivity against lipid peroxidation, reduction of non-specific protein adsorption, good stability of half-year electrical stimulation and beneficial ability against corrosion. The present study shows potential application of PANi
Acknowledgements
This study was supported by the National Natural Science Foundation of China (30870635, 31070868), and the National Program on Key Basic Research Projects of China (973 Program, 2005CB724306).
References (72)
- et al.
Electrical stimulation of isolated retina with microwire glass electrodes
J Neurosci Meth
(2004) - et al.
Polypyrrole doped with 2 peptide sequences from laminin
Biomaterials
(2006) - et al.
Electrical interfacing between neurons and electronics via vertically integrated sub-4 μm-diameter silicon probe arrays fabricated by vapor–liquid–solid growth
Biosens Bioelectron
(2010) - et al.
Cerebral astrocyte response to micromachined silicon implants
Exp Neurol
(1999) - et al.
Brain responses to micro-machined silicon devices
Brain res
(2003) - et al.
Preparation of PANI-coated poly(styrene-co-styrene sulfonate) nanoparticles in microemulsion media
Colloids and Surfaces A: Physicochemical and Engineering Aspects
(2009) - et al.
Ordered surfactant-templated poly(3, 4-ethylenedioxythiophene) (PEDOT) conducting polymer on microfabricated neural probes
Acta Biomater
(2005) - et al.
Conducting polymers in biomedical engineering
Prog Polym Sci
(2007) - et al.
Polyaniline coatings on aluminium alloy 6061–T6: Electrosynthesis and characterization
Electrochimica Acta
(2010) - et al.
Corrosion protection of aluminum and aluminum alloys by polyanilines: A potentiodynamic and photoelectron spectroscopy study
Synth Met
(1999)
Synthesis and characterization of electroactive and biodegradable ABA block copolymer of polylactide and aniline pentamer
Biomaterials
Electrical stimulation of excitable tissue: design of efficacious and safe protocols
J Neurosci Meth
Effects of small pulsed nanocurrents on cell viability in vitro and in vivo: Implications for biomedical electrodes
Biomaterials
Histopathologic evaluation of prolonged intracortical electrical stimulation
Exp Neurol
Pathology of damaging electrical stimulation in the retina
Exp Eye Res
Fe2+ and Fe3+ initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media
Chemistry and Physics of Lipids
Modifications of protein by polyunsaturated fatty acid ester peroxidation products
Biochim Biophy Acta
Formation of high-molecular-weight protein adducts by methyl docosahexaenoate peroxidation products
Biochim Biophy Acta
New insights into the retinal circulation: Inflammatory lipid mediators in ischemic retinopathy
Prostag Leukort Ess
Evidence for a free radical mechanism in aging and u.v.-irradiated ocular lenses
Exp Eye Res
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding
Anal Biochem
Polyaniline films with nanostructure used as neural probe coating surfaces
App Surf Sci
Protein adsorption under electrical stimulation of neural probe coated with polyaniline
Colloid Surface B: Biointerfaces
Sloane-Stanley, GH. A simple method for the isolation and purification of total lipides from animal tissues
J Biol Chem
The resolving effect of soybean storage protein subunits under different separation gel concentrations of SDS-PAGE
Chinese journal of oil crop sciences
Conducting polymers for neural interfaces: Challenges in developing an effective long-term implant
Biomaterials
Polypyrrole-coated electrospun PLGA nanofibers for neural tissue applications
Biomaterials
Polymerization of the conducting polymer poly(3, 4-ethylenedioxythiophene) (PEDOT) around living neural cells
Biomaterials
Conducting polymers as free radical scavengers
Synth Met
Enhancement of the growth of human endothelial cells by surface roughness at nanometer scale
Biomaterials
Grafting of poly(ethylene oxide) to the surface of polyaniline films through a chlorosulfonation method and the biocompatibility of the modified films
Colloid Interface Sci
Fe2+-induced lipid peroxidation kinetics in liposomes: The role of surface Fe2+ concentration in switching the reaction from acceleration to decay
Free Radic Biol Med
Modification of ion homeostasis by lipid peroxidation: roles in neuronal degeneration and adaptive plasticity
Trends Neurosci
Polyaniline as corrosion protection coatings on cold rolled steel
Polymer
Platinum electrode noise in the ENG spectrum
Med Biol Eng Comput
Biocompatibility considerations at stimulating electrode interfaces
Ann Biomed Eng
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