A facile method to prepare a versatile surface coating with fibrinolytic activity, vascular cell selectivity and antibacterial properties
Graphical abstract
Introduction
Thrombus formation on material surfaces remains the major problem that causes the failure of blood contacting devices. Multiple ways have been employed to prevent thrombosis: surface passivation; immobilization of bioactive agents; and surface endothelialization [1]. Surface passivation is to reduce non-specific protein adsorption to provide an anticoagulant function. Protein resistant surfaces are prepared by immobilizing hydrophilic polymers or zwitterionic polymers on the material surfaces. The bioactive components used to prepare bioactive surfaces include anticoagulants, antiplatelet agents and fibrinolytic agents. The function of anticoagulants and antiplatelet agents is to prevent thrombus formation. The fibrinolytic agents are used for destroying or breaking up a thrombus formed on the surface of blood contacting materials before it is able to cause damage. Surface endothelialization is to simulate the endothelium as an approach to hemocompatible materials. As the inner surface of natural blood vessels, the vascular endothelium that consists of endothelial cells provide the functions of regulating thrombosis, fibrinolysis, inflammation and angiogenesis [2]. As far as we know, endothelium is the only known surface that is truly blood compatible in all aspects. Although there are various approaches to prepare “thromboresistant” materials and “endothelium-like” material surfaces, no truly blood compatible artificial materials have yet been prepared. We have to admit that incorporation of only one function is not enough to develop artificial materials that have satisfactory antithrombogenicity, development of multifunctional materials is drawing increasing attention.
Dual-functional materials that have anti-fouling properties as well as a specific bioactive function have been investigated for many years [3]. Based on this idea, one can envisage co-immobilizing two or more bioactive components on the surface of the materials [4,5]. Recently, a single-molecule multipurpose modification strategy has been developed to construct multifunctional material surfaces [6]. The effect of various molecules such as heparin/heparin mimetics, gallic acid, various adhesion proteins and antibodies, and nitric oxide are used in the new strategies for developing material surfaces with multi-functions, including excellent hemocompatibility, inhibiting smooth muscle cell proliferation, and native endothelium regeneration. For example, surface heparinization has been predicted to be an effective approach to obtain outstanding biocompatibility and multifunction, including anticoagulant activity, inhibiting smooth muscle cell proliferation, and promoting endothelialization [7]. Heparin mimetics are usually synthetic polymers, for example biopolymer derivatives and synthetic sulfated/sulfonated/carboxylated artificial polymers with similar biological functions as heparin, including anticoagulant and growth factor binding properties [8]. Sulfonated polymers, e.g. poly(sodium 4-vinylbenzenesulfonate) (PSS), are one of the widely investigated heparin mimetics as surface modifier to prepare multifunctional material surfaces and are proven to improve anticoagulant properties, promote endothelialization, and inhibit smooth muscle cell proliferation [9,10].
Nevertheless, multifunctional modification is still immature. The multiple steps involved in the immobilization of different functional molecules are complex and lack controllability and repeatability. In the method of co-immobilization, the function of the previously introduced components may be compromised by the introduction of another function. The single-molecule multipurpose modification strategy is a promising way to obtain multifunctionalities but more molecules with multiple biofunctions needs to be explored to get more desirable biocompatibility. There is still a long way to go in designing materials that are multifunctional.
To overcome this problem, layer by layer (LbL) assembly technique based on electrostatic attraction between a positively and a negatively charged molecules was developed. LbL deposition is a convenient and substrate-independent method and has become one of the most promising surface modification strategies to prepare multifunctional blood and tissue contacting materials [11]. The main advantage of LBL technique is the ability to create stable deposited thin films with well-organized structure and tunable composition on different substrates [12]. It is reported that the deposited multilayers were stable for at least one week or even several weeks under physiological conditions [13,14]. Previous works have shown that chain segments of the previously adsorbed polyelectrolyte layer penetrate out of the outmost surface layer [15]. The extensive interpenetration of the layers facilitates the development of a multilayered coating surface with synergic properties of different functional molecules [[16], [17], [18]]. Moreover, the functional components will not interfere with each other, and these functions are tunable by manipulating the composition of the functional components in the outmost layer via altering assembly conditions [16].
Taking into account the advantages of LbL technique, we recently developed a new method for surface functionalization [[19], [20], [21]]. Specifically, a material’s surface is first coated with a multilayered polyelectrolyte film containing “guest” groups. This film further introduces “host” functional components via host-guest interactions. Previously, the simplicity, controllability and universality of this method that combines LbL techniques and host-guest chemistry have been fully explored in surface biofunctionalization [19]. However, the integration of multifunctional components and how to balance these functions on the material surface is still a challenging work.
In the present work, the gold substrate was first LbL deposited with a multilayered polyelectrolyte film containing chitosan (positively charged) and a copolymer of sodium 4-vinylbenzenesulfonate (SS) and the “guest” adamantane monomer 1-adamantan-1-ylmethyl methacrylate (P(SS-co-Ada), negatively charged) via electro-static interactions, referred to as Au-LBL. Then, “host” β-cyclodextrin derivatives bearing seven lysine ligands (CD-L) were immobilized on the Au-LBL surface by host-guest interactions (Au-LBL/CD-L). Chitosan was chosen as the cationic polyelectrolyte for LbL building because it is expected to impart good biocompatibility and antibacterial properties on surfaces. The component P(SS-co-Ada) is designed to impart the surface with heparin-like properties as well as facilitating incorporation of the “host” molecule CD-L. The incorporated CD-L is expected to lyse nascent clots on the surface. This strategy can not only dissolve nascent clots and avoid the neointimal hyperplasia but also enhance the ability of endothelialization and reduce the risk of biomaterial-associated infections after the implantation of biomedical materials, making the materials suitable for both short-term and long-term applications.
Section snippets
Materials
Silicon wafers (Guangzhou Semiconductor Material Research Institute, Guangzhou, China) were coated with gold (80 nm) by Suzhou Institute of Nano-Tech and Nano-Bionics (SINANO), Chinese Academy of Sciences, and were cut into 0.5 cm × 0.5 cm pieces. 4-vinylbenzenesulfonate (SS), β-cyclodextrin, fluorescein isothiocyanate labeled phalloidin (Phalloidin-FITC), 4′,6-diamidino-2-phenylindole (DAPI) and lysogeny broth (LB) were obtained from Sigma-Aldrich Chemical Company (USA). 2-mercaptoethylamine,
Preparation and characterization of Au-LbL/CD-L surfaces
The process of the preparation of Au-LbL/CD-L surfaces is shown in Scheme 1. Anionic polyelectrolyte P(SS-co-Ada) and cationic polyelectrolyte CHI were immobilized on the Au-NH2 surface through LbL. A 10 bilayer of polyelectrolyte film containing chitosan and P(SS-co-Ada) was then prepared. The component of PSS from the copolymer P(SS-co-Ada) is expected to impart heparin-mimicking properties on the surface [23]. The CHI component is predicted to resist bacteria attachment on the surface. The
Conclusions
In this work, a multifunctional material surface was developed by LbL deposition of positively charged chitosan and negatively charged P(SS-co-Ada), followed by incorporation of CD-L by host-guest interaction. The three components, chitosan, P(SS-co-Ada) and CD-L, provide a versatile surface coating with antibacterial properties, vascular cell selectivity and fibrinolytic activity (∼13 min to clot lysis). This strategy can not only dissolve nascent clots and avoid neointimal hyperplasia but
Declarations of interest
None.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2016YFC1100402), the National Natural Science Foundation of China (21774089 and 21334004), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
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