(524b) Quantifying Structure-Function Relationships of Protein-Selective Networks at the Micro- and Macro-Scale

Polymeric formulations and structures that act as high-affinity protein sorbents have a wide range of industrial and pharmaceutic applications. For example, high-affinity sorbents will aid in the separation of active therapeutic proteins from bioprocess byproducts via chromatography¹, but can also be applied in capsules and carriers to enhance the therapeutic’s loading capacity². Herein, we developed libraries of polymeric networks for high-affinity binding and maximal adsorption capacity of model cationic proteins. Simultaneous pursuit of affinity and loading is particularly interesting because the polymer properties that enhance each are paradoxical. By example, while hydrophobic moieties generally enhance protein-material affinity by enabling the formation of entropically favorable hydrophobic interactions, hydrophobicity decreases the polymer-solvent interaction parameter governing swelling (and loading capacity by association) in aqueous media. In this study, we present the synthesis and assessment of networks that possess a variety of composition and macromolecular structure, to understand this complex interplay and engineer useful protein sorbents.

The base network structure for all studies was a copolymer of methacrylic acid (30 mol%) methylenebisacrylamide (5 mol%) and acrylamide (remaining mol fractions). These networks were copolymerized with up to 40 mol% of either methyl methacrylate (MMA), tert-butyl methacrylate (tBMA), or benzyl methacrylate (BzMA) in substitution for some, but not all, of the acrylamide fraction. Microparticles of the above formulations were synthesized as films by UV-initiated free radical polymerization using Irgacure 184 as a photoinitiator, and manually crushed/sieved to yield a fine powder (<45 µm dry diameter). Nanoparticles of poly(acrylamide-co-methacrylic acid), which were necessary for many analyses, were synthesized by inverse emulsion polymerization as previously described³. For peptide-modification studies, pendant methacrylic acid moieties within nanoparticles were modified post-synthesis with a bifunctional amine-maleimide linker and a cysteine-containing peptide via sequential carbodiimide-coupling and thiol-maleimide Michael addition reactions. The composition of all copolymers were verified by FTIR, potentiometric titration (for methacrylic acid content) and a MicroBCA colorimetric assay (as appropriate, for peptide content). The morphology of microparticles was determined by electron microscopy, and the diameter of nanomaterials was determined by DLS.

Microparticles with a range of hydrophobic content were incubated individually with model proteins (i.e. lysozyme, cytochrome c, trypsin, bovine serum albumin, bovine hemoglobin, ovomuccoid) that possessed a range of molecular weight and isoelectric point. Adsorption of each species correlated strongly with the formation of ionic polymer-protein interactions, as evidenced by the substantial adsorption competition in high ionic strength buffers. Hydrophobic interactions, within certain constraints (up to 30% MMA, up to 20% tBMA, up to 10% BzMA) enhanced the adsorption of all high isoelectric point proteins to the microparticles, corresponding with the point of significant reduction in water content (equilibrium swelling studies). Hydrophobicity played a particularly major role in lysozyme adsorption, with optimal formulations loading 6.87-fold (30% MMA), 7.50-fold (10% tBMA), and 7.18-fold (5% BzMA) more lysozyme at saturation than formulations without hydrophobic monomer. Analysis of the solvent-accessible surface area (SASA) amino acid composition of each model protein lead to the conclusion that arginine and tryptophan residues (prevalent in lysozyme but not trypsin or cytochrome c) may be primarily responsible for lysozyme’s sensitivity to hydrophobic interactions, even in the presence of physiologic salt.

Protein-microparticle interaction studies, therefore, pointed to that small quantities (e.g. just 5 mol% of benzyl methacrylate) of hydrophobic moieties can optimize the affinity-permeability tradeoff and maximize protein partitioning while minimizing macroscopic effects on the network (i.e. water content, elastic modulus). To the aim of capitalizing on this optimum, while simultaneously enhancing single-protein network specificity, we conjugated hexamer peptides (identified previously^4,5) to poly(methacrylic acid-co-acrylamide) networks. These hexamers were optimized for trypsin adsorption, and led to enhancement of trypsin affinity, relative to unmodified controls as evidenced by equilibrium protein adsorption in quartz crystal microbalance with dissipation (QCM-D) studies.

In summary, an affinity-permeability tradeoff exists for hydrogels that contain hydrophobic moieties, as the formation of hydrophobic protein-polymer interactions and interaction of moiety-containing chains with solvent (water) must balance. Optimal levels of each hydrophobic monomer tested (MMA, tBMA, BzMA) were determined that did not deter hydrogel swelling in 1x phosphate buffered saline and enabled up to 7.5-fold enhancement in lysozyme adsorption. Further protein-specific adsorption enhancement was also explored for trypsin-peptide nanogel conjugates, following the affinity-permeability tradeoff guidelines established in microparticle studies. This study not only developed promising sorbents for lysozyme and trypsin, but through identifying a relevant thermodynamic tradeoff in gels will also inform the fabrication of high-affinity sorbents for alternate therapeutic proteins.

References:

[1] Xiu, L., Valeja, S. G., Alpert, A. J., Jin, S., & Ge, Y. (2014). Effective protein separation by coupling hydrophobic interaction and reverse phase chromatography for top-down proteomics. Analytical chemistry, 86(15), 7899-7906.

[2] Culver, H. R., Clegg, J. R., & Peppas, N. A. (2017). Analyte-responsive hydrogels: intelligent materials for biosensing and drug delivery. Accounts of chemical research, 50(2), 170-178.

[3] Zhong, J. X., Clegg, J. R., Ander, E. W., & Peppas, N. A. (2018). Tunable Poly ((methacrylic acid)‐co‐acrylamide) Nanoparticles through Inverse Emulsion Polymerization. Journal of Biomedical Materials Research Part A.

[4] Clegg JR, Gu J, Harger, M. and Peppas NA. Molecularly Imprinted Polymer-Peptide Hybrid Materials for the Recognition and Sequestration of Proteins. Biomedical Engineering Society Annual Meeting, Phoenix AZ. 10/14/17

[5] Clegg JR, Zhong JX, Ander EW, Sun JA, and Peppas NA. Versatile Anionic Nanogels for the Targeted Delivery of Therapeutic Small Molecules via Active and Passive Mechanisms. US-Japan Drug Delivery Symposium, Maui, HI. 12/16/17.