Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates

Abstract

The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.

Associative and Dissociative Processes in Non‐Covalent Polymer‐Mediated Intracellular Protein Delivery

“…Non-covalent polymer-protein binding studies, sometimes involvingd elivery experiments, is ab road field, and additional reports relatedt ot his topic that highlight various binding interactions and characterization methods can be found in Refs. [58][59][60][61][62][63][64][65][66][67][68][69][70][71].B inding studies showcasing availablet echniques could potentially contain criticali nformation necessary for future protein-delivery optimization studies and should not be overlooked.…”

Utilizing functionalized bromomaleimides for fluorogenic conjugation and PEGylation of enzymes

“…It is hypothesized that the bridging of disulfide bonds with a maleimide moiety could introduce additional stability, through preventing disulfide reduction. The study reported here investigated the formation of functionalized MAMs, ABMs and DTMs in enzyme conjugations and how the substituents affect the integrity of two enzymes, α‐chymotrypsin (α‐CT) and human lysozyme (HLZ), which are enzymes commonly used in conjugation‐based applications and have been extensively characterized …”

Ligands and characterization for effective bio‐atom‐transfer radical polymerization

“…Since the first studies of protein‐ATRP, protein–polymer conjugates with different polymer architectures and functionalities have been synthesized to manipulate the activity, stability, and substrate binding affinity of the conjugate. These previous studies indicated that to provide consistent and reliable performance, conjugates must be homogeneous in the number of chains attached and have low molar mass dispersities.…”