Nanoscale growth factor patterns by immobilization on a heparin-mimicking polymer

Abstract

In this study, electrostatic interactions between sulfonate groups of an immobilized polymer and the heparin binding domains of growth factors important in cell signaling were exploited to nanopattern the proteins. Poly(sodium 4-styrenesulfonate-co-poly(ethylene glycol) methacrylate) (pSS-co-pPEGMA) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using ethyl S-thiobenzoyl-2-thiopropionate as a chain transfer agent and 2,2'-azoisobutyronitrile (AIBN) as the initiator. The resulting polymer (1) was characterized by 1H NMR, GPC, FT-IR, and UV-vis and had a number average molecular weight (Mn) of 24,000 and a polydispersity index (PDI) of 1.17. The dithioester end group of 1 was reduced to the thiol, and the polymer was subsequently immobilized on a gold substrate. Binding of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) to the polymer via the heparin binding domains was then confirmed by surface plasmon resonance (SPR). The interactions were stable at physiological salt concentrations. Polymer 1 was cross-linked onto silicon wafers using an electron beam writer forming micro- and nanopatterns. Resolutions of 100 nm and arbitrary nanoscale features such as concentric circles and contiguous squares and triangles were achieved. Fluorescence microscopy confirmed that bFGF and VEGF were subsequently immobilized to the polymer micro- and nanopatterns.

Figure 1

Conjugation of proteins to nanopatterns of a heparin-mimicking polymer. a-b. Films of poly(styrene sulfonate-co-PEG methacrylate) 1 are exposed to electron beams to cross-link the polymer to the surface via radical coupling of the PEG side chains. c. VEGF and d. bFGF are conjugated to the surfaces via interaction of the heparin binding domains with the polymer. Protein representations obtained from the PDB (1VPF, 1BFG).

Scheme 1

Synthesis and reduction of pSS-co-PEGMA.

Figure 2

FT-IR spectra of a) PSS-co-PEGMA 1 powder and b) FT-IR spectrum of PSS-co-PEGMA 2 immobilized on a gold surface.

Figure 3

SPR analysis of growth factor binding to immobilized pSS-co-PEGMA. a. Injection of yellow line: 0.5 μg/mL, green line: 0.2 μg/mL, red line: 0.1μg/mL of bFGF, blue line: 0.2 μg/mL bFGF in the presence of 20 μg of heparin, and grey line: 0.2 μg/mL of control protein BSA. b. Injection of yellow line: 0.5 μg/mL, green line: 0.2 μg/mL, blue line: 0.1μg/mL of VEGF, and red line: 0.2 μg/mL VEGF in the presence of 20 μg of heparin. Solution: PBS containing 0.1% Triton X. Surfaces were regenerated with phosphate buffer containing 4 M NaCl. Protein representations were obtained from the PDB (1VPF, 1BFG).

Figure 4

Fluorescent images of a. bFGF and b. VEGF immobilized onto pSS-co-PEGMA micropatterns. VEGF and bFGF were visualized with antibody staining. Each patterned square is 5 micron by 5 micron in size.

Figure 5

a. Nanoscale patterns of pSS-co-PEGMA are visible in the height image taken with an atomic force microscope in tapping mode. Lines approximately 100 nm in width forming a square, triangle, concentric square and circle are observed. Fluorescent image of b. bFGF and c. VEGF bound to the nanopatterns with antibody staining. Scale bar = 5 μm.