(672g) Molecularly Imprinted Polymer-Peptide Hybrid Materials for Engineered Protein Recognition

The recognition of protein biomarkers is of utmost importance in medical applications including imaging, drug delivery, and regenerative medicine. Molecularly Imprinted Polymers (MIPs) are a class of biomimetic materials formed through the polymerization of functional monomers around a molecular template. Following template extraction, nanocavities possessing a complementary geometric and chemical profile to the template remain. Herein, we present the synthesis of MIPs on the surface of nanomaterials for drug delivery applications. We strengthen specific protein-MIP interactions through the synthetic incorporation of engineered oligopeptides, which have been selected through molecular docking simulation for high-affinity protein binding.

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.

[1] H. R. Culver, Stephanie D. Steichen, M. Herrera-Alonso, and Nicholas A. Peppas, â??A versatile route to colloidal stability and surface functionalization of hydrophobic nanomaterials,â?Under Review.

[2] S. Teletchea, J. Rey, V. Stresing, S. Hervouet, P. Tuffery, and D. Heymann, â??SolyPep: a fast generator of soluble peptides,â?In Preparation.

[3] M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray, and R. D. Taylor, â??Improved proteinâ??ligand docking using GOLD,â?Proteins Struct. Funct. Bioinforma., vol. 52, no. 4, pp. 609â??623, 2003.