Characterization of Molecularly Imprinted Particles Modified with Various Hydrophobic Monomers

Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. Particles were characterized for protein binding capacity to develop an optimal hydrogel composition for the oral delivery of a model protein therapeutic in response to binding free trypsin in the small intestine.

A displacement mechanism for protein delivery was verified equilibrating the hydrogels in solution with cytochrome c, the model therapeutic, before incubating the loaded hydrogels with trypsin, the target protein. The displacement mechanism showed more success with imprinted polymers than non-imprinted polymers. A poly(methacrylic acid-co-N,N-methylene bisacrylamide-co-acrylamide) (P(MAA-co-MBA-co-Aam)) composition was modified with hydrophobic monomers or peptides to increase affinity for the trypsin molecule and improve on the oral delivery mechanism.

Multiple formulations of hydrogels with various hydrophobic monomers in different proportions were synthesized using free radical polymerization. The successful incorporation of hydrophobic monomers into the microparticles was verified through Fourier Transform Infrared Spectroscopy (FTIR). Initially, in non-competitive solutions a composition substituted with 5 mol% benzyl methacrylate for acrylamide showed the greatest binding capacity for trypsin. However, in competitive solutions with other proteins present, adding hydrophobic monomer to a hydrogel decreased the specificity of the microparticles for trypsin. Thus, adding hydrophobic monomers increases the particles’ affinity for a protein in solution, but does not increase the specificity of the particles for a specific protein, trypsin.

Multiresponsive P(MAA-co-MBA-co-Aam) particles were synthesized and surface modified with peptide ligands. The peptide ligands were incorporated using EDC-NHS zero-length links between the carboxylic acid groups of the polymer and free primary amines on the peptide. The reaction of the peptide was confirmed by primary amine assays. Modifying the hydrogels with peptides specific for interactions with trypsin increased the competitiveness of the particles for trypsin when compared to unmodified hydrogels.

Ultimately, modifying the original P(MAA-co-MBA-co-Aam) structure with peptides increased the selectivity these particles had for trypsin, the target protein, while incorporating hydrophobic monomer into the polymer network decreased the selectivity the hydrogels had for trypsin.