Protein-polymer conjugates are important in diverse fields including drug delivery, biotechnology, and nanotechnology. This feature article highlights recent advances in the synthesis and application of protein-polymer conjugates by controlled radical polymerization techniques. Special emphasis on new applications of the materials, particularly in biomedicine, are highlighted.
Constructing multicomponent protein structures that match the complexity of those found in Nature is essential for the next generation of medical materials. In this report, a versatile method to precisely arrange multicomponent protein nanopatterns in two-dimensional single-layer or three-dimensional multilayer formats using electron beam lithography is described. Eight arm poly(ethylene glycol)s were modified at the chain ends with either biotin, maleimide, aminooxy, or nitrilotriacetic acid. Analysis by 1H NMR spectroscopy revealed that the reactions were efficient and that end group conversions were 91-100%. The polymers were then cross-linked onto Si surfaces using electron beams to form micron sized patterns of the functional groups. Proteins with biotin binding sites, a free cysteine, an N-terminal α-oxoamide, and a histidine tag, respectively, were then incubated with the substrate in aqueous solutions without the addition of any other reagents. By fluorescence microscopy experiments it was determined that proteins reacted site-specifically with the exposed functional groups to form protein micropatterns. Multicomponent nanoscale protein patterns were then fabricated. Different PEGs with orthogonal reactivity were sequentially patterned on the same chip. Simultaneous assembly of two different proteins from a mixture of the biomolecules formed the multicomponent two dimensional patterns. Atomic force microscopy demonstrated that nanometer sized patterns of polymer were formed and fluorescence microscopy demonstrated that side-by-side patterns of the different proteins were obtained. Moreover, multilayer PEG fabrication produced micron and nanometer sized patterns of one functional group on top of the other. Precise three-dimensional arrangements of different proteins were then realized.
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 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 micron- and nano- patterns. 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 nano- patterns.
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