Electropolymerized bioresistant coatings of OEGylated dendron carbazoles: Design parameters and protein resistance SPR studies

Abstract

The electrografting of oligoethylene glycol or (OEG)ylated carbazole linear dendrons and their protein adsorption resistance properties have been investigated by surface plasmon resonance (SPR) spectroscopy. A series of the carbazole dendron generations, G₀, G₁, G₂ were synthesized and electrodeposited by cyclic voltammetry (CV) on Au-glass substrate which also served as a surface for evanescent waveguide excitation in SPR. In addition, the films were characterized by in-situ electrochemical SPR (EC-SPR), static water contact angle, and X-ray photoelectron spectroscopy (XPS) measurements. It was observed that electropolymerized films prepared from the higher generation linear-dendron G₂, is most effective in preventing non-specific protein adsorption as observed by SPR kinetic measurements using fibrinogen as model protein. Film thickness also played a critical role in protein adsorption resistance - electrodeposition approaching monolayer thickness gave the highest protein resistance. In addition, the films were evaluated for non-specific protein binding against the smaller proteins, lysozyme and bovine serum albumin (BSA). The study provides insight to manipulating the architecture and composition of protein resistant materials deposited on metals and semi-conducting substrates and their possible use in biomedical applications.

Introduction

Investigating the physical adsorption of proteins onto solid substrates is a key step for evaluating the performance of biomedical devices, biosensors, and bio-microfluidic systems. Non-specific binding can reduce the functionality of an interface that is potentially useful in bio-recognition or bioimplant devices, i.e. by fouling or producing high background noise and “false positives”. In tissue contacting applications, non-specific protein adsorption may promote the adhesion of macrophages and other cells, leading to inflammatory and foreign body responses. For devices in contact with blood, even small quantities of adsorbed protein may initiate coagulation as well as platelet adhesion and activation, leading ultimately to thrombus formation. The biofouling of surfaces initiated by the random, non-specific adsorption of proteins can result in the loss of useful lifespan or efficacy for prostheses, implanted biosensors, and contact lenses; decreased accuracy and detection level of protein and DNA arrays in drug discovery/clinical diagnoses; exogenous species invasion in isolated ecosystems; and in industrial applications, increased costs for maintenance and corrosion mitigation [1], [2], [3], [4]. Preventing non-specific protein adsorption is thus of critical importance in the design of surfaces and of significant value to biotechnology, health care, energy, and marine industries.

For anti-thrombogenic implants, three types of materials, including heparin, poly(ethylene glycol) (PEG), and phospholipids, have been commonly employed for surface modification with polymers or self-assembled monolayers (SAMs) [5], [6], [7]. Biomaterial surfaces modified with heparin have become a major coating technique commercially to prevent thrombin activation of existing materials. However, this approach lacks long-term stability for implantation. Recently, the use of lipid films to prevent protein adsorption is seen as a potential alternative to other materials. On the outer surface of a cell, zwitterionic lipid phosphorylcholine (PC) is a major component and is known to be non-thrombogenic [7]. Methacryloyloxyethyl phosphorylcholine (MPC)-based copolymers have become one of the major synthetic biocompatible materials [8]. However, such films can be unstable in solution in the presence of albumin and are prone to detachment when passed through an air/water interface [9], [10]. A well-established strategy to improve the biocompatibility of a surface is grafting poly(ethylene glycol) (PEG) [11], [12], [13], [14], [15]. PEG-based materials are commonly used to effectively resist non-specific protein adsorption even with long-range properties. However, PEG is a polyether that can autoxidize, especially in the presence of oxygen and transition metal ions found in most biochemically relevant solutions [12]. Still, its relative success as a protein-resistant coating, as well as the number of literature on the biological interaction of PEG, have made it the gold standard for most new studies of non-fouling surfaces.

Conducting polymers (CP) or π-conjugated polymers have been extensively studied during the last 30 years for their fundamental properties and potential applications [16]. This includes: semiconductor devices, electrical capacitors, sensors, anodes for fuel cells, corrosion protection, though they have not been specifically targeted for bio-resistant surfaces. A major drawback of CPs as coatings is their poor morphology with direct deposition by electropolymerization of heteroaromatic electroactive monomers.Recently, we have demonstrated the deposition of high optical quality ultrathin film coatings of conjugated polymers through the precursor polymer approach [17]. We have reported the synthesis and electrochemical deposition of “passive” polysiloxane–precursor derivatives (by cyclic voltammetry or potentiostatic methods) of thiophene and pyrrole to form cross-linked polythiophene and polypyrrole, respectively [18], [19]. On the other hand, it is also possible to synthesize “active” precursor polymers in which the polymer backbone itself is conjugated and shows electro-optical activity [17]. These polymers can be directly deposited from an electrolyte solution or spin-coated to the electrode substrate and then cross-linked electrochemically. Poly(vinyl carbazole) or PVK as precursor polymer for cross-linked polycarbazole thin films for electro-optical device applications has been investigated [20]. The electroactive carbazole side group facilitated electrodeposition forming oligocarbazole networks. In addition, we have reported the use of carbazole bearing dendrimers and dendrons that can be electrodeposited on electrode surfaces [21], [22]. The electro-optical properties and electrodeposition of these dendrimers and dendrons were found to be generation dependent. Different protein resistant materials are typically applied onto a desired surface via physical adsorption, SAMs, chemical grafting or surface initiated polymerization methods, though the use of electrochemical polymerization techniques is relatively unexplored [11], [23], [24], [25]. In this study, we have introduced the OEGylated carbazole linear dendron molecules that could be electrochemically deposited on different metal or electrode substrates suitable for biomedical applications.

Oligo(ethylene glycol) or OEG as short as three repeating units in helical or amorphous forms has also been shown to resist non-specific protein adsorption similar to PEGs [11]. Understanding the molecular mechanisms leading to the PEG’s protein resistance has also gained much attention since different molecular mechanisms may be at work [11], [12], [13], [14], [15], [16], [17]. Prime and Whitesides demonstrated that both hydroxyl- and methoxy-terminated OEG SAMs on Au begins to exhibit this resistance at 35% surface coverage [11]. They illustrated that the presence of flexible OEG strands is necessary for protein resistance. This phenomenon is explained by the prevention of direct interaction between the surface and the protein by forming a stable solid–liquid interface involving tightly bound water. Quantum calculations by Grünze also suggested that the densely-packed all-trans phase of PEG terminated SAM cannot form a stable solvation layer, while the helical structure stabilizes such a layer [13]. On the other hand, Szleifer showed that the presence of a dense and inert film prevents contact between the substrate and the protein rendering the film protein-resistant [14]. From the above theories, it is clear that grafting density and the intrinsic hydration of PEG determines protein resistance. Recently, however, Hess argued that the distribution of the PEG polymer chains is closer to random as opposed to the assumptions of the previous theories that the polymer chains are evenly distributed across the surface, with a constant spacing determined by the grafting density [15]. Over the years, the aforementioned systematic studies on protein adsorption have been done on model surfaces like Au, however, for practical applications, various substrates including metal, metal alloys, and semi-conducting materials are widely used for both medical and non-medical applications [26]. Thus, the ability to manipulate the architecture and composition of a protein resistant material that can be deposited on other electrically conducting (electrode) substrates is necessary in order to create more widely applicable coatings.

Dendrimers and dendrons (fractional dendrimers) are highly branched, monodispersed macromolecules with a well-defined three dimensional and globular structure with potential applications in the field of chemical and biomedical sensors, microelectronic and biomimetic systems, adhesion, coating, and membrane chemistry, and nanotechnology [27], [28], [29], [30]. The ability to control the generation, the type of branching, the reactivity and number of focal and peripheral functionality, and composition make them attractive for modifying interfaces [31]. Baker et al have utilized dendrons as spacers to control the space around individual probe molecules [32]. For bio-interfaces, one possibility of applying the dendrimer architecture is to use self-assembled layers or layer-by-layer films of dendrimers with subsequent functionalization with PEG macromolecules. Andronov et al have showed that protein adsorption increased upon surface dendronization with aliphatic polyester dendrons supporting previous studies indicating that chain mobility and dynamics in water may be critical to protein repulsion [36]. Another architecture is to introduce a dendronized moiety on OEG chains to produce linear-dendron materials. In this case, OEG chains can be attached to the dendron focal point were peripheral functionality remain reactive. Haag and co-workers have investigated the chemisorption of monothiolated glycerol dendrons with different sizes and functionalities and reported the optimal size (number of generation) necessary to inhibit the adsorption of the proteins through a better and more defined monolayer [34].

In this study, non-specific protein adsorption resistant surfaces were fabricated from OEGylated dendron macromolecules with the carbazole moiety capable of electrochemical polymerization on different substrates suitable for biomedical applications (Fig. 1).

The aim is to present an alternative and yet versatile approach in fabricating a bioinert surface that offer nanoscale control over the thin film architecture and thickness. By introducing OEGylated carbazole dendron macromolecules, it should be possible to generate electrically conducting surfaces primarily through electropolymerization of the carbazole units – to form π-conjugated polycarbazole films. Three different generations of OEGylated carbazole dendrons, G₀CbztEG, G₁CbztEG, and G₂CbztEG, were synthesized and electrochemically deposited onto Au surfaces and evaluated for their protein resistance using surface plasmon resonance (SPR) spectroscopy. Fibrinogen was used as a model protein. Also, the conditions for electrodeposition were optimized and the films were tested against smaller proteins such as bovine serum albumin (BSA) and lysozyme under buffered conditions. The results are discussed in terms of deposited film properties, linear dendron architectures, and film thickness.