Multilayer hydrogel coatings to combine hemocompatibility and antimicrobial activity
Introduction
Bacterial infections related to indwelling medical devices rank as the fifth-leading cause of hospital patients death in the US [4]. Central venous catheter-associated bloodstream infections occur with a frequency of 5 per 1000 catheter days and result in a lethality rate of 20% [5]. Therefore, in addition to sterile handling and systemic administration of anticoagulants, antimicrobial prevention is of paramount importance for the safety of central venous catheter applications [6]. Current strategies to locally prevent infections include bactericidal and bacteriostatic approaches using antiseptically impregnated coatings or antibiotic lock therapies. For that purpose, both the catheter bulk material and hydrophilic hydrogel coatings, providing catheters with a “slippery-when-wet” lubricious surface, were customized to embed and release bioactives with silver being the most frequently applied bioactive component. In these approaches, silver is applied as silver bulk material, in ionic form or as silver nanoparticles (AgNPs) referring to both oxidation states (Ag0/Ag+). Although the antimicrobial mechanism of silver is still controversially discussed [7], [8], [9], [10], [11], [12], [13], there both oxidation states are considered to be active against bacteria, most probably through a conversion of bacterial sulfhydryl (R-SH) groups [9]. For silver ions, the biological activity is multifold including the inhibition of cell respiration and the inactivation of enzymes to effects on DNA replication and cell division [7], [8].
A common strategy for sustained Ag+ release is the reduction of ionic silver in solution to form AgNPs with a large surface-to-volume ratio [14]. Previous studies have shown that AgNPs can either penetrate bacterial cells [10] or become deposited on the bacterial cell wall, affecting membrane permeability as well as electrolyte and metabolite transport [11]. The cytotoxicity of silver nanoparticles results from interactions with membrane proteins, the activation of signaling pathways and the inhibition of cell proliferation [12], [13]. The toxicity of silver for bacterial and eukaryotic cells has been extensively tested and compared in earlier studies, showing a significantly lower toxicity of silver for the eukaryotic cells [15], [16].
The use of silver-loaded hydrogel coatings for central venous catheters dates back to 1998 [2] and was found suitable to prevent early-onset catheter-related infections but concluded to be limited for long lasting protection [1], [2]. Recently reported strategies have exploited the in situ formation of AgNPs within swollen hydrogel networks of catheter coatings [17], [18], [19] which were developed to minimize protein adsorption and platelet adhesion [18]. However, the antiseptic functionalization of biomaterials with silver-based compounds often is associated with a loss of the beneficial hemocompatibility characteristics since silver not only affects microorganisms, but also can exert undesired side effects on mammalian cells [20], [21]. Thus, biomaterial coatings combining sustained, long-term delivery of antimicrobial active silver and non-thrombogenic properties are particularly important targets of current research [22].
Herein, we present a new concept to address these challenges. A recently introduced star-shaped poly(ethylene glycol) (PEG)-heparin hydrogel [23] was grafted as a thin film coating onto thermoplastic polyurethane bulk materials and further modified with silver by incubation in AgNO3 solution (see Scheme 1). The building blocks, star-shaped PEG and heparin, widely applied in the surface modification of biomaterials due to their protein-resisting and anticoagulant characteristics, were covalently linked to form a hydrogel network. In an effort to further modulate this system, a multilayer gel coating was developed, where a silver loaded hydrogel is coated onto the surface of the polyurethane bulk material with a second silver-free hydrogel layer on top to act as a diffusion barrier for prolonged silver release and for avoiding direct cellular interactions with AgNPs at the coating-blood interface. These multilayer hydrogel coatings were thoroughly evaluated in whole blood hemocompatibility assays for coagulation and inflammatory activation. The antiseptic capacity of the coating was verified with different relevant bacterial strains.
Section snippets
Preparation of starPEG-heparin hydrogel coatings
Biohybrid starPEG-heparin gels were prepared using amino functionalized four-armed starPEG and EDC-s-NHS activated heparin as previously described [3]. Briefly, a solution of heparin (14 000 g/mol; Calbiochem, Darmstadt, Germany) was activated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) (ratio 2:1) at a stoichiometric balanced concentration of sulfo-NHS to the starPEG–NH2–groups. Subsequently, a starPEG solution (10 000 g/mol Polymer
Characterization of hydrogels and hydrogel coatings on TPU
Hydrogel immobilization to TPU substrates requires modification of the TPU surface to increase the number of available reactive groups for the substrate-gel coupling reaction. Different TPU surface modification techniques were evaluated, amongst them oxygen and ammonia plasma and electron beam treatment in nitrogen atmosphere and under air conditions. Electron beam treatment of TPU induced an increase in O/C ratio as detected by XPS (data not shown). The C1s peak of the TPU can be deconvoluted
Discussion
Combining antimicrobial and anticoagulant properties within biomaterial coatings defines a practically important challenge of current research. With the reported approach, we were able to merge two previously established principles, namely the antimicrobial properties of AgNPs and the anticoagulant potential of heparin-containing biohybrid hydrogels within a multifunctional biomaterial.
Conclusion
Multilayered silver-functionalized starPEG-heparin hydrogels combining antiseptic and hemocompatible properties have been developed as a versatile surface coating to fight the infection of blood contacting medical products such as central venous catheters. The introduced multilayer architecture was demonstrated to balance hemocompatibility and antiseptic efficacy. Specifically, a silver-free starPEG-heparin layer on top of an AgNP containing starPEG-heparin layer resulted in a hemocompatible
Acknowledgment
We thank Monique Marx (preparation of surfaces and hemocompatibility assays) and Martina Franke (gold evaporation) for technical assistance. We furthermore gratefully acknowledge contributions by Dr. Heike Hund (ITM, TU Dresden) and Dr. Petr Formanek (IPF Dresden) for performing AAS and SEM analyses, respectively. We thank in particular Dr. Mirko Nitschke for surface plasma treatments, XPS measurements and the related scientific discussions. We are thankful for the contributions of Dr. Elke
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