Romantic surfaces : Zwitterionic polymer brushes for biomedical applications

Abstract

The non-specific adsorption of biomolecules, often referred to as fouling, is a recurring issue in many biomedical and bioanalytical applications. For example, the non-specific adsorption of proteins diminishes the signal-to-noise ratio of biosensing systems, while protein coronas formed on drug delivery vehicles hamper the drug to reach its target site due to fast removal by phagocytic cells. Zwitterionic polymer brushes have been brought forward as excellent candidates to prevent fouling. This thesis provides tools and insights to improve antifouling performance in general, to efficiently introduce functionality to zwitterionic polymer brushes and to transfer existing knowledge of antifouling coatings on flat surfaces onto microbeads. In addition, two applications for the antifouling zwitterionic polymer-coated microbeads are also presented. Chapter 1 provides an introduction to antifouling coatings in general and to zwitterionic polymer brushes in particular. This chapter also introduces the concept of a romantic surface: a surface that repels all biomolecules except that special one it is interested in. Chapter 2 describes a novel approach on how to create such romantic surfaces. An azide-functionalized sulfobetaine is introduced that enables the 3D-functionalization of fully zwitterionic polymer brushes. The number of built-in functional groups could be tuned by the copolymerization of this novel azido-SB monomer with a standard sulfobetaine. The incorporated azide moieties can then be used to couple recognition units of choice using “click” chemistry. Functionalization of the brushes with biotin units enabled the specific binding of avidin without the interference of the non-specific binding of fibrinogen. Particles of various sizes are being used for a wide range of biomedical application, including drug delivery, imaging, cells sorting and as biosensing platform. As on flat surfaces, fouling compromises the performance of these particles. Chapter 3 describes a methodology to efficiently coat microbeads with zwitterionic antifouling coatings. Top functionalization of the polymer brush chain ends was used to introduce various functional groups, including biotin and mannose moieties. The beads were able to specifically bind streptavidin and ConA, respectively, in the presence of a 10 % serum solution. Flow cytometry was shown to be a powerful tool to evaluate both non-specific as well as specific binding of proteins in a quick and automated manner. The methodology developed in chapter 3 was used for chapters 4, 5 and 6. Chapter 4 presents a systematic comparison of various antifouling polymer brushes to deepen the understanding on the exact relationship between monomer structure and antifouling performance. Sulfobetaines with three methylene groups between opposite charges (SB-3) have often been compared to carboxybetaines with two separating methylene groups (CB-2). In chapter 4, for the first time, a direct comparison was made between SB-3, CB-2 and SB-2 (a sulfobetaine with two separating methylene groups), revealing that both carbon spacer length as well as the nature of the charged groups have an influence on the antifouling capacity. A phosphoscholine and a hydroxyl acrylamide monomer were also shown to produce excellent antifouling surfaces. It was also demonstrated that the bead-based platform, as described in chapter 3, provides a great tool to evaluate antifouling performance. Immunoprecipitation followed by mass spectrometry (IP-MS) is a powerful technique to study protein-protein interactions. The differentiation between true protein interactors and proteins that bind non-specifically is, however, often challenging. Chapter 5 describes the use of zwitterionic polymer brush-coated microbeads to reduce background signals in IP-MS experiments. The azide-functionalized sulfobetaine monomer that was introduced in chapter 2, was used to enable the immobilization of antibodies via click chemistry. Antifouling anti-GFP beads were produced, which showed similar levels of GFP capture as commercially available anti-GFP beads, but with strongly reduced non-specific protein binding. In an IP-MS experiment many contaminating proteins were identified with the commercially available anti-GFP beads, whereas the antifouling anti-GFP beads were able to almost exclusively purify the GFP-tagged bait protein and its known interaction partners. The non-specific adsorption of proteins onto intravenous injected particles often leads to the rapid clearance from the blood stream by phagocytic cells like macrophages and other antigen-presenting cells. This drastically hampers the in vivo performance of the particles. Chapter 6 describes the use of antifouling zwitterionic polymer brush-coated microbeads to prevent phagocytosis by murine macrophages. The functionalization of the antifouling beads with proteins did not result in a significant increase in phagocytic uptake. On the other hand, when the beads were specifically targeted to the macrophages by the immobilization of IgG2a antibodies, the beads were actively internalized via Fc receptor-mediated phagocytosis. This chapter demonstrates the high potential of functionalized zwitterionic polymer brush-coated particles for in vitro and in vivo applications. Chapter 7 provides a general discussion on the most important achievements that are outlined within this thesis. Special attention is given to suggestions for future research. Novel monomers are proposed that may lead to improved antifouling capacity. In addition, potential applications of antifouling beads are discussed.