(672g) Molecularly Imprinted Polymer-Peptide Hybrid Materials for Engineered Protein Recognition
Polycaprolactone (PCL) nanoparticles were formed by solvent displacement methods using a custom polymaleic anhydride-alt-1-octadecene-g-polyethylene glycol methacrylate (PMAO-g-PEGMA) surfactant according to the method developed by Culver et al1. PCL nanoparticles were selected in particular because of their biodegradable nature and ability to encapsulate a hydrophobic therapeutic agent. Imprinted and control non-imprinted polymers (NIPs) were synthesized through the polymerization of anionic, cationic, and hydrophilic functional monomers with 5% crosslinker and PMAO-g-PEGMA functionalized PCL in 0.1x PBS, in the presence (MIPs) or absence (NIPs) of 0.5 mg/mL trypsin, a model high isoelectric point protein template.
Oligopeptides which dock in high affinity with trypsin were selected through a systematic molecular docking study. One thousand random, water-soluble 6-mer peptides were selected using the soly-pep random sequence generator available online from Paris Diderot University2. These peptides were docked to an isolated volume of trypsin using GOLD3, defined as a spherical volume centered at the active site. 28 promising sequences, which docked in high affinity with trypsin, lacked arginine and lysine residues, and contained a cysteine for thiol-coupling were isolated. Oligopeptide selectivity for trypsin was quantified through a systematic surface plasmon resonance (SPR) study. Oligopeptides were conjugated to dextran coated SPR sensors through thiol-coupling in order to mimic their orientation while coupled to MIP networks. The ligand-coated sensor was then sequentially exposed to trypsin, lysozyme, cytochrome c, and hemoglobin at a range of concentrations while transient protein adhesion was monitored.
MIPs in the absence of oligopeptides bound 59±22% more trypsin than corresponding NIPs across a range of solution concentrations up to 0.5 mg/mL. These MIPs also bound 48±17% more lysozyme and 47±22% more cytochrome c, which are high isoelectric point proteins with a smaller hydrodynamic diameter than trypsin. Hemoglobin, which is a low isoelectric point protein with a larger hydrodynamic diameter than trypsin, was excluded significantly from MIPs as compared to NIPs. Oligopeptides, which possessed micromolar affinity for trypsin as validated by SPR, were incorporated at 88% efficiency into MIPs and NIPs through a thiol coupling reaction. These peptide-MIP hybrid materials, which exhibit recognitive properties for trypsin, can serve as a model system for the development of future intelligent theranostic hydrogels.
This work was supported in part by the UT-Portugal Collaborative Research Program (CoLab). JRC is supported by an NSF Graduate Research Fellowship.
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