Synthetic Semiflexible and Bioactive Brushes

Abstract

Polymer brushes are extensively used for the preparation of bioactive surfaces. They form a platform to attach functional (bio)molecules and control the physicochemical properties of the surface. These brushes are nearly exclusively prepared from flexible polymers, even though much stiffer brushes from semiflexible polymers are frequently found in nature, which exert bioactive functions that are out of reach for flexible brushes. Synthetic semiflexible polymers, however, are very rare. Here, we use polyisocyanopeptides (PICs) to prepare high-density semiflexible brushes on different substrate geometries. For bioconjugation, we developed routes with two orthogonal click reactions, based on the strain-promoted azide–alkyne cycloaddition reaction and the (photoactivated) tetrazole–ene cycloaddition reaction. We found that for high brush densities, multiple bonds between the polymer and the substrate are necessary, which was achieved in a block copolymer strategy. Whether the desired biomolecules are conjugated to the PIC polymer before or after brush formation depends on the dimensions and required densities of the biomolecules and the curvature of the substrate. In either case, we provide mild, aqueous, and highly modular reaction strategies, which make PICs a versatile addition to the toolbox for generating semiflexible bioactive polymer brush surfaces.

Semiflexible Immunobrushes Induce Enhanced T Cell Activation and Expansion

“…To synthesize T cell activating PIC immunobrushes, we applied a methodology we recently developed to graft PIC polymers to various surfaces. 41 Here, we aimed to synthesize PIC block copolymers containing orthogonal functional groups to introduce T cell activating signals on one side of the polymer and use the other side to attach the polymers to 4.5 μm magnetic beads ( Figure 1 a). The first block contained allyl functional groups, which were subsequently modified with tetrazole-biotin via photoinduced nitrile imine-mediated tetrazole-ene cycloaddition (NITEC) reaction.…”

“…Furthermore, we have shown before that PICs can be easily coupled to different types of scaffolds such as nanoparticles or flat surfaces. 41 Finally, other types of flexible and semiflexible polymers could be used as brushes to investigate the effect of polymer stiffness on T cell activation.…”

“…End-functional PIC were synthesized according to the previously reported protocol. 41 Briefly, PICs with an allyl containing first block were prepared. For this, methoxy-, allyl-, and azide-terminated monomers were prepared as described according to the literature.…”

“… 37 The allyl functionalities in the first block were then converted to biotin via the nitrile imine-mediated tetrazole-ene cycloaddition (NITEC) reaction using a tetrazole-biotin compound as described before. 41 Biotin conjugation was confirmed by measuring the fluorescence of the pyrazolines that are formed upon conjugation using a SPARK M10 plate reader from TECAN. The biotin end-functional PIC was then dissolved at 2 mg/mL in PBS and labeled overnight at 4 °C with dibenzocyclooctyne (DBCO)-conjugated Alexa Fluor 594 dye (Click Chemistry Tools) using a 1:500 ratio of dye/azide and used without purification.…”

“…The beads were incubated with the desired biotin compounds on a rotator overnight at 4 °C and washed with 2× 0.05% PBS Tween and 3× with PBS. The beads were analyzed by flow cytometry on a BD FACS Verse cytometer and via the previously reported stripping assay, 41 with the exception that as a stripping solution, 3 mM biotin in 2% sodium dodecyl sulfate (SDS) in Milli-Q water was used. For the beads made for the antibody density series, only flow cytometry analysis was performed to determine antibody densities and PIC densities from the Ab/PIC ratio determined after polymer purification…”