(97e) Anionic Microgels for the Trypsin-Mediated Retention and Release of Therapeutic Proteins

Protein therapeutics are currently administered by intravenous, subcutaneous, or intramuscular injection. While injection provides an effective administration route, the discomfort it entails causes low rates of patient compliance. Thus, delivering protein therapeutics orally is more desirable and will promote patient compliance. In order to achieve oral protein delivery, the polymeric carrier needs to be able to withstand harsh gastric conditions to protect the fragile protein cargo, and have a targeted release mechanism at the site of the small intestine.

In this study, several polymer systems were designed for the oral delivery of a high isoelectric point protein therapeutic in response to binding free trypsin that is abundantly present in the small intestine.

Previous studies had shown that a displacement mechanism using complementary ionic interactions between the microparticles and model therapeutic proteins in solution was viable. A displacement mechanism for protein delivery was verified by loading the hydrogels with cytochrome c, a model high isoelectric point therapeutic, before incubating the loaded hydrogels with trypsin, the target protein. The amount of cytochrome c released and trypsin adsorbed were quantified. Results showed higher binding capacity in low ionic strength solutions than high ionic strength solutions, indicating that the release mechanism was predominated by ionic interactions, and not by specific affinity for the trypsin molecule.

To increase our system’s affinity for trypsin, the base polymer was modified with various hydrophobic monomers. Additionally, the base particles were surface modified with specific peptide ligands.

A base polymer with methacrylic acid, N,N-methylene bisacrylamide, and acrylamide was modified with hydrophobic monomers or peptides to increase affinity for the trypsin molecule and efficiently release cytochrome c in response to the presence of trypsin. Multiple formulations of hydrogels with various hydrophobic monomers in different proportions (i.e. 5, 10, 20, and 40 mol%) were synthesized using free radical polymerization. The successful incorporation of hydrophobic monomers into the microparticles was verified through Fourier Transform Infrared Spectroscopy (FTIR) by comparing characteristic amide, methacrylate, and aromatic peaks.

The base particles were synthesized and also surface modified with peptide ligands. The peptide ligands were incorporated using EDC-NHS bifunctional linker between the carboxylic acid groups of the polymer and free primary amines on the peptide. The coupling of the peptide was verified by detecting primary amines via fluorescamine assay and microBCA colorimetric assay.

The binding capacities of these particles for model proteins were assessed and compared to particles with hydrophobic monomer. While particles incorporated with hydrophobic monomers decreased binding capacity of the particles for cytochrome c, modifying microparticles with oligopeptide ligands increased the specificity that the particles had for cytochrome c and trypsin.

This study meant to design polymer systems for the oral delivery of high isoelectric point protein therapeutics in response to the presence of trypsin. Two platforms, one with varying proportions of hydrophobic monomer, and one with peptide ligands modified onto the surface, were synthesized. The binding capacity of these platforms for a model therapeutic, cytochrome c, and for trypsin, were quantified. Particles surface modified with peptide ligands showed increased specificity for cytochrome c and trypsin when compared to the unmodified controls.